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LibertadDigital \ 2015 





Garland Science 

Taylor&Francis Group 


LibertadDigital \ 2015 

Garland Science 

Vice President: Denise Schanck 
Sénior Editor: Michael Morales 

Production Editor and Layout: Emma Jeffcock of EJ Publishing 

Illustrator: Nigel Orme 
Developmental Editor: Monica Toledo 
Editorial Assistants: Lamia Harik and Alina Yurova 
Copy Editor: Jo Clayton 

Book Design: Matthew McClements, Blink Studio, Ltd. 

Cover Illustration: José Ortega 

Authors Album Cover: Photography, Christophe Carlinet; 
Design, Nigel Orme 
Indexer: Bill Johncocks 

Uploaded to LibertadDigital by Giordano Bruno (2015) 

© 2014 by Bruce Alberts, Dennis Bray, Karen Hopkin, 
Alexander Johnson, Julián Lewis, Martin Raff, Keith Roberts, 
and Peter Walter 

© 2010 by Bruce Alberts, Dennis Bray, Karen Hopkin, 
Alexander Johnson, Julián Lewis, Martin Raff, Keith Roberts, 
and Peter Walter 

© 2004 by Bruce Alberts, Dennis Bray, Karen Hopkin, 
Alexander Johnson, Julián Lewis, Martin Raff, Keith Roberts, 
and Peter Walter 

© 1998 by Bruce Alberts, Dennis Bray, Alexander Johnson, 
Julián Lewis, Martin Raff, Keith Roberts, and Peter Walter 

This book contains information obtained from authentic and 
highly regarded sources. Every effort has been made to trace 
copyright holders and to obtain their permission for the use of 
copyright material. Reprinted material is quoted with permis¬ 
sion, and sources are indicated. A wide variety of references are 
listed. Reasonable efforts have been made to publish reliable 
data and information, but the author and the publisher cannot 
assume responsibility for the validity of all materials or for the 
consequences of their use. 

All rights reserved. No part of this book covered by the copy¬ 
right hereon may be reproduced or used in any format in any 
form or by any means—graphic, electronic, or mechanical, in- 
cluding photocopying, recording, taping, or information storage 
and retrieval systems—without permission of the publisher. 

ISBNs: 978-0-8153-4454-4 (hardcover); 978-0-8153-4455-1 

Published by Garland Science, Taylor & Francis Group, LLC, 
an informa business, 711 Third Avenue, New York, NY 10017, 
USA, and 3 Park Square, Milton Park, Abingdon, OX14 4RN, UK. 

Printed in the United States of America 
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 

Essential Cell Biology Website 

Artistic and Scientific Direction: Peter Walter 

Narrated by: Julie Theriot 

Producen Michael Morales 

About the Authors 

Bruce Alberts received his PhD from Harvard University 
and is the Chancellor's Leadership Chair in Biochemistiy 
and Biophysics for Science and Education, University of 
California, San Francisco. He was the editor-in-chief of 
Science magazine from 2008-2013, and for twelve years 
he served as President of the U.S. National Academy of 
Sciences (1993-2005). 

Dennis Bray received his PhD from Massachusetts Institute 
of Technology and is currently an active emeritus professor 
at the University of Cambridge. 

Karen Hopkin received her PhD in biochemistry from 
the Albert Einstein College of Medicine and is a Science 
writer in Somerville, Massachusetts. She is a contributor to 
ScientificAmerican's daily podcast, 60-Second Science, and to 
E. O. Wilson's digital biology textbook, Ufe on Earth. 
Alexander Johnson received his PhD from Harvard 
University and is Professor of Microbiology and Immunology 
at the University of California, San Francisco. 

Julián Lewis received his DPhil from the University of 
Oxford and is an Emeritus Scientist at the London Research 
Institute of Cáncer Research UK. 

Martin Raff received his MD from McGill University and is 
at the Medical Research Council Laboratory for Molecular 
Cell Biology and Cell Biology Unit at University College 

Keith Roberts received his PhD from the University of 
Cambridge and was Deputy Director of the John Innes 
Centre, Norwich. He is currently Emeritus Professor at the 
University of East Anglia. 

Peter Walter received his PhD from The Rockefeller 
University in New York and is Professor of the Department 
of Biochemistry and Biophysics at the University of 
California, San Francisco, and an Investigator of the Howard 
Hughes Medical Institute. 

Library of Congress Cataloging-in-Publication Data 

Alberts, Bruce. 

Essential cell biology / Bruce Alberts [and seven others]. 
— Fourth edition. 
pages cm. 

ISBN 978-0-8153-4454-4 (hardback) 

1. Cytology. 2. Molecular biology. 3. Biochemistry. I. Title. 
QH581.2.E78 2013 



Garland Science 

Taylor & Francis Group 

Vislt our website at 

LibertadDigital \ 2015 


In our world there is no form of matter more astonishing than the liv- 
ing cell: tiny, fragüe, marvelously intricate, continually made afresh, yet 
preserving in its DNA a record of information dating back more than 
three billion years, to a time when our planet had barely cooled frorn 
the hot materials of the nascent solar system. Ceaselessly re-engineered 
and diversified by evolution, extraordinarily versatile and adaptable, the 
cell retains a complex core of self-replicating Chemical machineiy that is 
shared and endlessly repeated by every living organism on the face of the 
Earth—in every animal, every leaf, every bacterium in a piece of cheese, 
every yeast in a vat of wine. 

Curiosity, if nothing else, should drive us to study cell biology; we need to 
understand cell biology to understand ourselves. But there are practical 
reasons, too, why cell biology should be a part of everyone's education. 
We are made of cells, we feed on cells, and our world is made habit¬ 
able by cells. The challenge for scientists is to deepen our knowledge of 
cells and find new ways to apply it. All of us, as citizens, need to know 
something of the subject to grapple with the modem world, from our 
own health affairs to the great public issues of environmental change, 
biomedical technologies, agriculture, and epidemic disease. 

Cell biology is a big subject, and it has links with almost every other branch 
of Science. The study of cell biology therefore provides a great scientific 
education. However, as the Science advances, it becomes increasingly 
easy to become lost in detail, distracted by an overload of information 
and technical terminology. In this book we therefore focus on providing 
a digestible, straightforward, and engaging account of only the essential 
principies. We seek to explain, in a way that can be understood even by 
a reader approaching biology for the first time, how the living cell works: 
to show how the molecules of the cell—especially the protein, DNA, and 
RNA molecules—cooperate to create this remarkable system that feeds, 
responds to stimuli, moves, grows, divides, and duplicates itself. 

The need for a clear account of the essentials of cell biology became 
apparent to us while we were writing Molecular Biology of the Cell (MBoC), 
now in its fifth edition. MBoC is a large book aimed at advanced under- 
graduates and gradúate students specializing in the life Sciences or 
medicine. Many students and educated lay people who require an intro- 
ductory account of cell biology would find MBoC too detailed for their 
needs. Essential Cell Biology (ECB), in contrast, is designed to provide the 
fundamentáis of cell biology that are required by anyone to understand 
both the biomedical and the broader biological issues that affect our lives. 
This fourth edition has been extensively revised. We have brought every 
part of the book up to date, with new material on regulatory RNAs, 
induced pluripotent stem cells, cell suicide and reprogramming, the 
human genome, and even Neanderthal DNA. In response to student 
feedback, we have improved our discussions of photosynthesis and DNA 

LibertadDigital \ 2015 

repair. We have added many new figures and have updated our cover- 
age of many exciting new experimental techniques—including RNAi, 
optogenetics, the applications of new DNA sequencing technologies, and 
the use of mutant organisms to probe the defects underlying human dis- 
ease. At the same time, our "How We Know" sections continué to present 
experimental data and design, illustrating with specific examples how 
biologists tackle important questions and how their experimental results 
shape future ideas. 

As before, the diagrams in ECB emphasize central concepts and are 
stripped of unnecessary details. The key terms introduced in each chapter 
are highlighted when they first appear and are collected together at the 
end of the book in a large, illustrated glossary. 

A central feature of the book is the many questions that are presented in 
the text margins and at the end of each chapter. These are designed to 
provoke students to think carefully about what they have read, encourag- 
ing them to pause and test their understanding. Many questions challenge 
the student to place the newly acquired information in a broader biologi- 
cal context, and some have more than one valid answer. Others invite 
speculation. Answers to all the questions are given at the end of the book; 
in many cases these provide a commentaiy or an alternative perspective 
on material presented in the main text. 

For those who want to develop their active grasp of cell biology further, 
we recommend Molecular Biology of the Cell, Fifth Edition: A Problems 
Approach, by John Wilson and Tim Hunt. Though written as a compan- 
ion to MBoC, this book contains questions at all levels of difficulty and 
contains a goldmine of thought-provoking problems for teachers and 
students. We have drawn upon it for some of the questions in ECB, and 
we are veiy grateful to its authors. 

The explosión of new imaging and Computer technologies continúes 
to provide fresh and spectacular views of the inner workings of living 
cells. We have captured some of this excitement in the new Essential Cell 
Biology website, located at This 
site, which is freely available to anyone in the world with an interest in 
cell biology, contains over 150 video clips, animations, molecular struc- 
tures, and high-resolution micrographs—all designed to complement the 
material in individual book chapters. One cannot watch cells crawling, 
dividing, segregating their chromosomes, or rearranging their surface 
without a sense of wonder at the molecular mechanisms that underlie 
these processes. For a vivid sense of the marvel that Science reveáis, it 
is hard to match the narrated movie of DNA replication. These resources 
have been carefully designed to make the leaming of cell biology both 
easier and more rewarding. 

Those who seek references for further reading will find them on the ECB 
student and instructor websites. But for the very latest reviews in the cur- 
rent literature, we suggest the use of web-based search engines, such as 
PubMed ( ) or Google Scholar ( 
As with MBoC, each chapter of ECB is the product of a communal 
effort, with individual drafts circulating frorn one author to another. In 
addition, many people have helped us, and these are credited in the 
Acknowledgments that follow. Despite our best efforts, it is inevitable 
that there will be errors in the book. We encourage readers who find them 
to let us know at, so that we can correct these 
errors in the next printing. 

LibertadDigital \ 2015 


The authors acknowledge the many contributions of 
professors and students from around the world in the 
creation of this fourth edition. In particular, we are grate- 
ful to the students who participated in our focus groups; 
they provided invaluable feedback about their experi- 
ences using the book and our multimedia, and many of 
their suggestions were implemented in this edition. 

We would also like to thank the professors who helped 
organize the student focus groups at their schools: 
Nancy W. Kleckner at Bates College, Kate Wright and 
Dina Newman at Rochester Institute of Technology, 
David L. Gard at University of Utah, and Chris Brandl 
and Derek McLachlin at University of Western Ontario. 
We greatly appreciate their hospitality and the opportu- 
nity to learn from their students. 

We also received detailed reviews from many instruc- 
tors who used the third edition, and we would like to 
thank them for their contributions: Devavani Chatterjea, 
Macalester College; Frank Hauser, University of 
Copenhagen; Alan Jones, University of North Carolina at 
Chapel Hill; Eugene Mesco, Savannah State University; 
M. Scott Shell, University of California Santa Barbara; 
Grith Lykke Sorensen, University of Southern Denmark; 
Marta Bechtel, James Madison University; David 
Bourgaize, Whittier College; John Stephen Horton, 
Union College; Sieirn Lim, Nanyang Technological 
University; Satoru Kenneth Nishimoto, University of 
Tennessee Health Science Center; Maureen Peters, 
Oberlin College; Johanna Rees, University of Cambridge; 
Gregg Whitworth, Grinnell College; Karl Fath, Queens 
College, City University of New York; Barbara Frank, 
Idaho State University; Sarah Lundin-Schiller, Austin 
Peay State University; Marianna Patrauchan, Oklahoma 
State University; Ellen Rosenberg, University of British 
Columbia; Leslie Kate Wright, Rochester Institute of 
Technology; Steven H. Denison, Eckerd College; David 
Featherstone, University of Illinois at Chicago; Andor 
Kiss, Miami University; Julie Lively, Sewanee, The 
University of the South; Matthew Rainbow, Antelope 
Valley College; Juliet Spencer, University of San Francisco; 
Christoph Winkler, National University of Singapore; 
Richard Bird, Auburn University; David Burgess, Boston 

College; Elisabeth Cox, State University of New York, 
College at Geneseo; David L. Gard, University of Utah; 
Beatrice Holton, University of Wisconsin Oshkosh; Glenn 
H. Kageyama, California State Polytechnic University, 
Pomona; Jane R. Dunlevy, University of North Dakota; 
Matthias Falk, Lehigh University. We also want to thank 
James Hadfield of Cáncer Research UK Cambridge 
Institute for his review of the methods chapter. 

Special thanks go to David Morgan, a coauthor of MBoC, 
for his help on the signaling and cell división chapters. 

We are very grateful, too, to the readers who alerted us 
to errors they had found in the previous edition. 

Many staff at Garland Science contributed to the crea¬ 
tion of this book and made our work on it a pleasure. 
First of all, we owe a special debt to Michael Morales, 
our editor, who coordinated the whole enterprise. He 
organized the initial reviewing and the focus groups, 
worked closely with the authors on their chapters, 
urged us on when we fell behind, and played a major 
part in the design, assembly, and production of Essential 
Cell Biology student website. Monica Toledo managed 
the flow of chapters through the book development 
and production process, and oversaw the writing of 
the accompanying question bank. Lamia Harik gave 
editorial assistance. Nigel Orme took original draw- 
ings created by author Keith Roberts and redrew them 
on a Computer, or occasionally by hand, with great 
skill and flair. To Matt McClements goes the credit for 
the graphic design of the book and the creation of the 
chapter-opener sculptures. As in previous editions, 
Emma Jeffcock did a brilliant Job in laying out the whole 
book and meticulously incorporating our endless cor- 
rections. Adam Sendroff and Lucy Brodie gathered user 
feedback and launched the book into the wide world. 
Denise Schanck, the Vice President of Garland Science, 
attended all of our writing retreats and orchestrated 
everything with great taste and diplomacy. We give our 
thanks to everyone in this long list. 

Last but not least, we are grateful, yet again, to our col- 
leagues and our families for their unflagging tolerance 
and support. 

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LibertadDigital \ 2015 

Resources for Instructors and Students 

The teaching and learning resources for instructors and 
students are available online. The instructor’s resources 
are password protected and available only to quali- 
fied instructors. The student resources are available to 
everyone. We hope these resources will enhance student 
learning, and make it easier for instructors to prepare 
dynamic lectures and activities for the classroom. 


Instructor Resources are available on the Garland 
Science Instructor's Resource Site, located at www. The website provides 
access not only to the teaching resources for this book 
but also to all other Garland Science textbooks. Qualified 
instructors can obtain access to the site from their sales 
representative or by emailing 

Art of Essential Cell Biology, Fourth Edition 

The images from the book are available in two conven- 
ient formats: PowerPoint® and JPEG. They have been 
optimized for display on a Computer. Figures are search- 
able by figure number, figure ñame, or by keywords used 
in the figure legend from the book. 

Figure-lntegrated Lecture Outlines 

The section headings, concept headings, and figures 
from the text have been integrated into PowerPoint 
presentations. These will be useful for instructors who 
would like a head start creating lectures for their course. 
Like all of our PowerPoint presentations, the lecture 
outlines can be customized. For example, the content 
of these presentations can be combined with videos and 
questions from the book or "Question Bank," in order to 
create unique lectures that facilítate interactive learning. 

Animations and Videos 

The 130+ animations and videos that are available to 
students are also available on the Instructor's Resource 
site in two formats. The WMV-formatted movies are 
created for instructors who wish to use the movies in 
PowerPoint presentations on Windows® computers; the 
QuickTime-formatted movies are for use in PowerPoint 
for Apple computers or Keynote® presentations. The 
movies can easily be downloaded to your Computer 
using the "download" button on the movie preview page. 

Question Bank 

Written by Linda Huang, University of Massachusetts, 
Boston, and Cheiyl D. Vaughan, Harvard University 
División of Continuing Education, the revised and 
expanded question bank ineludes a variety of question 
formats: múltiple choice, fill-in-the-blank, true-false, 
matching, essay, and challenging "thought" questions. 
There are approximately 60-70 questions per chapter, 
and a large number of the multiple-choice questions 
will be suitable for use with personal response Systems 
(that is, clickers). The Question Bank was created with 
the philosophy that a good exam should do much more 
than simply test students' ability to memorize informa- 
tion; it should require them to reflect upon and intégrate 
information as a part of a sound understanding. It pro¬ 
vides a comprehensive sampling of questions that can 
be used either directly or as inspiration for instructors to 
write their own test questions. 


Adapted from the detailed references of Molecular 
Biology of the Cell, and organized by the table of con- 
tents for Essential Cell Biology, the "References" provide 
a rich compendium of journal and review articles for ref- 
erence and reading assignments. The "References" PDF 
document is available on both the instructor and student 

Medical Topics Guide 

This document highlights medically relevant topics cov- 
ered throughout the book, and will be particularly useful 
for instructors with a large number of premedical, health 
Science, or nursing students. 

Media Guide 

This document overviews the multimedia available for 
students and instructors and contains the text of the 
voice-over narration for all of the movies. 

Blackboard® and LMS Integration 

The movies, book images, and student assessments that 
accompany the book can be integrated into Blackboard 
or other learning management Systems. These resources 
are bundled into a "Common Cartridge" that facilitates 
bulk uploading of textbook resources into Blackboard and 
other learning management systems. The LMS Common 
Cartridge can be obtained on a DVD from your sales rep¬ 
resentative or by emailing 

LibertadDigital \ 2015 

x Resources for Instructors and Students 


The resources for students are available on the Essential 
Cell Biology Student Website, located at www.garland 
Science. com/ECB4-studen ts. 

Animations and Videos 

There are over 130 movies, covering a wide range of cell 
biology topics, which review key concepts in the book 
and illuminate the cellular microcosm. 

Student Self-Assessments 

The website contains a variety of self-assessment tools 
to help students. 

• Each chapter has a multiple-choice quiz to test 
basic reading comprehension. 

• There are also a number of media assessments that 
require students to respond to specific questions 
about movies on the website or figures in the book. 

• Additional concept questions complement the 
questions available in the book. 

• "Challenge" questions are included that provide a 
more experimental perspective or require a greater 
depth of conceptual understanding. 

Cell Explorer 

This application teaches cell morphology through inter- 
active micrographs that highlight important cellular 


Each chapter contains a set of flashcards, built into the 
website, that allow students to review key terms frorn 
the text. 


The complete glossary from the book is available on the 
website and can be searched or browsed. 


A set of references is available for each chapter for fur- 
ther reading and exploration. 

LibertadDigital \ 2015 

Contents and Special Features 

Chapter 1 Cells: The Fundamental Units of Life 1 

Panel 1-1 Microscopy 10-11 

Panel 1-2 Cell Architecture 25 

How We Know: Life's Common Mechanisms 30-31 

Chapter 2 Chemical Components of Cells 39 

How We Know: What Are Macromolecules? 60-61 

Panel 2-1 Chemical Bonds and Groups 66-67 

Panel 2-2 The Chemical Properties of Water 68-69 

Panel 2-3 An Outline of Some of the Types of Sugars 70-71 

Panel 2-4 Fatty Acids and Other Lipids 72-73 

Panel 2-5 The 20 Amino Acids Found ¡n Proteins 74-75 

Panel 2-6 A Survey of the Nucleotides 76-77 

Panel 2-7 The Principal Types of Weak Noncovalent Bonds 78-79 

Chapter 3 Energy, Catalysis, and Biosynthesis 83 

Panel 3-1 Free Energy and Biological Reactions 96-97 

How We Know: Measuring Enzyme Performance 104-106 

Chapter 4 Protein Structure and Function 121 

Panel 4-1 A Few Examples of Some General Protein Functions 122 

Panel 4-2 Making and Using Antibodies 146-147 

How We Know: Probing Protein Structure 162-163 

Panel 4-3 Cell Breakage and Initial Fractionation of Cell Extracts 164-165 

Panel 4-4 Protein Separation by Chromatography 166 

Panel 4-5 Protein Separation by Electrophoresis 167 

Chapter 5 DNA and Chromosomes 171 

How We Know: Genes Are Made of DNA 174-176 

Chapter 6 DNA Replication, Repair, and Recombination 197 

How We Know: The Nature of Replication 200-202 

Chapter 7 From DNA to Protein: How Cells Read the Genome 223 

How We Know: Cracking the Genetic Code 240-241 

Chapter 8 Control of Gene Expression 261 

How We Know: Gene Regulation—the Story of Eve 274-275 

Chapter 9 How Genes and Genomes Evolve 289 

How We Know: Counting Genes 316-317 

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Contents and Special Features 

Chapter 10 Modern Recombinant DNA Technology 

How We Know: Sequencing The Human Genome 



Chapter 11 Membrane Structure 

HowWe Know: Measuring Membrane Flow 



Chapter 12 Transport Across Cell Membranes 

How We Know: Squid Reveal Secrets of Membrane Excitability 



Chapter 13 How Cells Obtain Energy From Food 

Panel 13-1 Details of the 10 Steps of Glycolysis 

Panel 13-2 The Complete Citric Acid Cycle 

How We Know: Unraveling the Citric Acid Cycle 





Chapter 14 Energy Generation in Mitochondria and Chloroplasts 

How We Know: How Chemiosmotic Coupling Drives ATP Synthesis 

Panel 14-1 Redox Potentials 




Chapter 15 Intracellular Compartments and Protein Transport 

How We Know: Tracking Protein and Vesicle Transport 



Chapter 16 Cell Signaling 

HowWe Know: Untangling Cell Signaling Pathways 



Chapter 17 Cytoskeleton 

How We Know: Pursuing Microtubule-Associated Motor Proteins 



Chapter 18 The Cell-Division Cycle 

How We Know: Discovery of Cyclins and Cdks 

Panel 18-1 The Principal Stages of M Phase in an Animal Cell 




Chapter 19 Sexual Reproduction and the Power of Genetics 

Panel 19-1 Some Essentials of Classical Genetics 

How We Know: Using SNPs To Get a Handle on Human Disease 




Chapter 20 Cell Communities: Tissues, Stem Cells, and Cáncer 

How We Know: Making Sense of the Genes That Are Critical for Cáncer 



LibertadDigital \ 2015 

Detailed Contents 


Cells: The Fundamental Units of Ufe 

Cells Vary Enormously ¡n Appearance and Function 
Living Cells All Have a Similar Basic Chemistry 
All Present-Day Cells Have Apparently Evolved 
from the Same Ancestral Cell 
Genes Provide the Instructions for Cell Form, 
Function, and Complex Behavior 

The Invention of the Light Microscope Led to the 
Discovery of Cells 

Light Microscopes Allow Examination of Cells 
and Some of Their Components 
The Fine Structure of a Cell Is Revealed by 
Electron Microscopy 

Prokaryotes Are the Most Diverse and Numerous 
Cells on Earth 

The World of Prokaryotes Is Divided into Two 
Domains: Bacteria and Archaea 

The Nucleus Is the Information Store of the Cell 
Mitochondria Generate Usable Energy from 
Food to Power the Cell 
Chloroplasts Capture Energy from Sunlight 
Internal Membranes Create Intracellular 
Compartments with Different Functions 
The Cytosol Is a Concentrated Aqueous Gel 
of Large and Small Molecules 
The Cytoskeleton Is Responsible for Directed 
Cell Movements 

The Cytoplasm Is Far from Static 
Eukaryotic Cells May Have Originated as 


Molecular Biologists Have Focused on E. coli 
Brewer's Yeast Is a Simple Eukaryotic Cell 
Arabidopsis Has Been Chosen as a Model Plant 
Model Animáis Inelude Flies, Fish, Worms, 
and Mice 

Biologists Also Directly Study Human Beings 
and Their Cells 

Comparing Genome Sequences Reveáis Life's 

Common Heritage 33 

Genomes Contain More Than Just Genes 35 

Essential Concepts 35 

Questions 37 

Chapter 2 

Chemical Components of Cells 39 


Cells Are Made of Relatively Few Types of Atoms 40 
The Outermost Electrons Determine How Atoms 
Internet 41 

Covalent Bonds Form by the Sharing of Electrons 44 
There Are Different Types of Covalent Bonds 45 

Covalent Bonds Vary in Strength 46 

lonic Bonds Form by the Gain and Loss of 

Electrons 46 

Noncovalent Bonds Help Bring Molecules 

Together in Cells 47 

Hydrogen Bonds Are Important Noncovalent 

Bonds For Many Biological Molecules 48 

Some Polar Molecules Form Acids and Bases 

in Water 49 


A Cell Is Formed from Carbón Compounds 50 

Cells Contain Four Major Families of Small 

Organic Molecules 51 

Sugars Are Both Energy Sources and Subunits 

of Polysaccharides 52 

Fatty Acid Chains Are Components of Cell 

Membranes 53 

Amino Acids Are the Subunits of Proteins 55 

Nucleotides Are the Subunits of DNA and RNA 56 

Each Macromolecule Contains a Specific 

Sequence of Subunits 59 

Noncovalent Bonds Specify the Precise Shape 

of a Macromolecule 62 

Noncovalent Bonds Allow a Macromolecule 

to Bind Other Selected Molecules 63 

Essential Concepts 64 

Questions 80 





























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Detailed Contents 

Chapter 3 

Energy, Catalysis, and Biosynthesis 

Biological Order Is Made Possible by the 
Release of Heat Energy from Cells 
Cells Can Convert Energy from One Form to 

Photosynthetic Organisms Use Sunlight to 
Synthesize Organic Molecules 
Cells Obtain Energy by the Oxidation of 
Organic Molecules 

Oxidation and Reduction Involve Electron 

Chemical Reactions Proceed in the Direction 
that Causes a Loss of Free Energy 
Enzymes Reduce the Energy Needed to Initiate 
Spontaneous Reactions 
The Free-Energy Change for a Reaction 
Determines Whether It Can Occur 
AG Changes As a Reaction Proceeds Toward 

The Standard Free-Energy Change, AG°, Makes 
it Possible to Compare the Energetics of 
Different Reactions 

The Equilibrium Constant Is Directly Proportional 
to AG° 

In Complex Reactions, the Equilibrium Constant 
Ineludes the Concentrations of All Reactants 
and Products 

The Equilibrium Constant Indicates the 
Strength of Molecular Interactions 
For Sequential Reactions, the Changes in 
Free Energy Are Additive 
Thermal Motion Allows Enzymes to Find Their 

V max and Km Measure Enzyme Performance 
The Formation of an Activated Carrier Is 
Coupled to an Energetically Favorable 

ATP Is the Most Widely Used Activated Carrier 
Energy Stored in ATP Is Often Harnessed to 
Join Two Molecules Together 
NADH and NADPH Are Both Activated 
Carriers of Electrons 

NADPH and NADH Have Different Roles in Cells 
Cells Make Use of Many Other Activated 

The Synthesis of Biological Polymers Requires 
an Energy Input 

Essential Concepts 

Chapter 4 

Protein Structure and Function 121 


The Shape of a Protein Is Specified by Its Amino 
Acid Sequence 123 

Proteins Fold into a Conformation of Lowest 

Energy 126 

Proteins Come in a Wide Variety of Complicated 
Shapes 127 

The a Helix and the p Sheet Are Common 

Folding Patterns 130 

Hélices Form Readily in Biological Structures 130 

P Sheets Form Rigid Structures at the Core 

of Many Proteins 132 

Proteins Have Several Levels of Organizaron 132 

Many Proteins Also Contain Unstructured 

Regions 134 

Few of the Many Possible Polypeptide Chains 

Will Be Useful 135 

Proteins Can Be Classified into Families 136 

Large Protein Molecules Often Contain More 

Than One Polypeptide Chain 137 

Proteins Can Assemble into Filaments, Sheets, 

or Spheres 138 

Some Types of Proteins Have Elongated Fibrous 
Shapes 139 

Extracellular Proteins Are Often Stabilized by 

Covalent Cross-Linkages 140 


All Proteins Bind to Other Molecules 141 

There Are Billions of Different Antibodies, 

Each with a Different Binding Site 143 

Enzymes Are Powerful and Highly Specific 

Catalysts 144 

Lysozyme lllustrates How an Enzyme Works 145 

Many Drugs Inhibit Enzymes 149 

Tightly Bound Small Molecules Add Extra 

Functions to Proteins 149 


The Catalytic Activities of Enzymes Are Often 

Regulated by Other Molecules 151 

Allosteric Enzymes Have Two or More Binding 

Sites That Influence One Another 151 

Phosphorylation Can Control Protein Activity 

by Causing a Conformational Change 152 

Covalent Modifications Also Control the 

Location and Interaction of Proteins 154 

GTP-Binding Proteins Are Also Regulated by the 
Cyclic Gain and Loss of a Phosphate Group 155 
ATP Hydrolysis Allows Motor Proteins to 

Produce Directed Movements in Cells 155 

Proteins Often Form Large Complexes That 

Function as Protein Machines 156 






























LibertadDigital \ 2015 

Detailed Contents 


Proteins Can be Purified from Cells or Tissues 157 
Determining a Protein's Structure Begins with 

Determining Its Amino Acid Sequence 158 

Genetic Engineering Techniques Permit the 
Large-Scale Production, Design, and Analysis 
of Almost Any Protein 160 

The Relatedness of Proteins Aids the Prediction 
of Protein Structure and Function 161 

Essential Concepts 168 

Questions 169 

Chapter 5 DNA and Chromosomes 171 


A DNA Molecule Consists of Two Complementary 
Chains of Nucleotides 173 

The Structure of DNA Provides a Mechanism 

for Heredity 178 


Eukaryotic DNA Is Packaged into Múltiple 

Chromosomes 179 

Chromosomes Contain Long Strings of Genes 180 
Specialized DNA Sequences Are Required for 
DNA Replication and Chromosome 
Segregation 182 

Interphase Chromosomes Are Not Randomly 

Distributed Within the Nucleus 183 

The DNA in Chromosomes Is Always Highly 

Condensed 184 

Nucleosomes Are the Basic Units of Eukaryotic 

Chromosome Structure 185 

Chromosome Packing Occurs on Múltiple Levels 187 

Changes in Nucleosome Structure Allow 

Access to DNA 188 

Interphase Chromosomes Contain Both 
Condensed and More Extended Forms 
ofChromatin 190 

Essential Concepts 192 

Questions 193 

Chapter 6 DNA Replication, Repair, 

and Recombination 197 


Base-Pairing Enables DNA Replication 198 

DNA Synthesis Begins at Replication Origins 199 

Two Replication Forks Form at Each Replication 

Origin 199 

DNA Polymerase Synthesizes DNA Using a 

Parental Strand as Témplate 203 

The Replication Fork Is Asymmetrical 204 

DNA Polymerase Is Self-correcting 205 

Short Lengths of RNA Act as Primers for 

DNA Synthesis 206 

Proteins at a Replication Fork Cooperate to 

Form a Replication Machine 207 

Telomerase Replicates the Ends of Eukaryotic 

Chromosomes 209 


DNA Damage Occurs Continually in Cells 212 

Cells Possess a Variety of Mechanisms for 

Repairing DNA 213 

A DNA Mismatch Repair System Removes 

Replication Errors That Escape Proofreading 214 
Double-Strand DNA Breaks Require a Different 

Strategy for Repair 215 

Homologous Recombination Can Flawlessly 

Repair DNA Double-Strand Breaks 216 

Failure to Repair DNA Damage Can Have Severe 
Consequences for a Cell or Organism 218 

A Record of the Fidelity of DNA Replication and 
Repair Is Preserved in Genome Sequences 219 

Essential Concepts 220 

Questions 221 

Chapter 7 From DNA to Protein: 

How Cells Read the Genome 223 


Portions of DNA Sequence Are Transcribed 

into RNA 225 

Transcription Produces RNA That Is 

Complementary to One Strand of DNA 226 

Cells Produce Various Types of RNA 227 

Signáis in DNATell RNA Polymerase Where 

to Start and Finish Transcription 228 

Initiation of Eukaryotic Gene Transcription 

Is a Complex Process 230 

Eukaryotic RNA Polymerase Requires General 

Transcription Factors 231 

Eukaryotic mRNAs Are Processed in the Nucleus 232 
In Eukaryotes, Protein-Coding Genes Are 
Interrupted by Noncoding Sequences 
Called Introns 233 

Introns Are Removed From Pre-mRNAs by 

RNA Splicing 234 

Mature Eukaryotic mRNAs Are Exported 

from the Nucleus 236 

mRNA Molecules Are Eventually Degraded 

in the Cytosol 237 

The Earliest Cells May Have Had Introns in 

Their Genes 237 


An mRNA Sequence Is Decoded in Sets of 

Three Nucleotides 239 

tRNA Molecules Match Amino Acids to 

Codons in mRNA 242 

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Specific Enzymes Couple tRNAs to the Correct 
Amino Acid 

The mRNA Message Is Decoded by Ribosomes 
The Ribosome Is a Ribozyme 
Specific Codons in mRNA Signal the Ribosome 
Where to Start and to Stop Protein Synthesis 
Proteins Are Made on Polyribosomes 
Inhibitors of Prokaryotic Protein Synthesis Are 
Used as Antibiotics 

Controlled Protein Breakdown Helps Regúlate 
the Amount of Each Protein in a Cell 
There Are Many Steps Between DNA and 

Life Requires Autocatalysis 
RNA Can Both Store Information and Catalyze 
Chemical Reactions 

RNA Is Thought to Predate DNA in Evolution 

Essential Concepts 


Chapter 8 Control of Gene Expression 

The Different Cell Types of a Multicellular 
Organism Contain the Same DNA 
Different Cell Types Produce Different Sets 
of Proteins 

A Cell Can Change the Expression of Its Genes 
in Responseto External Signáis 
Gene Expression Can Be Regulated at Various 
Steps from DNA to RNA to Protein 

Transcription Regulators Bind to Regulatory 
DNA Sequences 

Transcriptional Switches Allow Cells to Respond 
to Changes in Their Environment 
Repressors Turn Genes Off and Activators 
Turn Them On 

An Activator and a Repressor Control the Lac 

Eukaryotic Transcription Regulators Control 
Gene Expression from a Distance 
Eukaryotic Transcription Regulators Help 
Initiate Transcription by Recruiting 
Chromatin-Modifying Proteins 

Eukaryotic Genes Are Controlled by 

Combinations of Transcription Regulators 
The Expression of Different Genes Can Be 
Coordinated by a Single Protein 
Combinatorial Control Can Also Generate 
Different Cell Types 

Specialized Cell Types Can Be Experimentally 
Reprogrammed to Become Pluripotent 
Stem Cells 278 

The Formation of an Entire Organ Can Be 

Triggered by a Single Transcription Regulator 278 
Epigenetic Mechanisms Allow Differentiated 

Cells to Maintain Their Identity 279 


Each mRNA Controls Its Own Degradation and 
Translation 281 

Regulatory RNAs Control the Expression of 

Thousands of Genes 282 

MicroRNAs Direct the Destruction of Target 

mRNAs 282 

Small Interfering RNAs Are Produced From 
Double-Stranded, Foreign RNAs to Protect 
Cells From Infections 283 

Thousands of Long Noncoding RNAs May Also 

Regúlate Mammalian Gene Activity 284 

Essential Concepts 284 

Questions 286 

Chapter 9 How Genes and Genomes 
Evolve 289 


In Sexually Reproducing Organisms, Only 
Changes to the Germ Line Are Passed 
On To Progeny 291 

Point Mutations Are Caused by Failures of the 
Normal Mechanisms for Copying and 
Repairing DNA 293 

Point Mutations Can Change the Regulation 

of a Gene 294 

DNA Duplications Give Rise to Families of 

Related Genes 294 

The Evolution of the Globin Gene Family 

Shows How Gene Duplication and Divergence 
Can Produce New Proteins 296 

Whole-Genome Duplications Have Shaped the 

Evolutionary History of Many Species 298 

Novel Genes Can Be Created by Exon 

Shuffling 298 

The Evolution of Genomes Has Been 
Profoundly Influenced by the Movement 
of Mobile Genetic Elements 299 

Genes Can Be Exchanged Between Organisms 

by Horizontal Gene Transfer 300 


Genetic Changes That Provide a Selective 

Advantage Are Likely to Be Preserved 301 

Closely Related Organisms Have Genomes 
That Are Similar in Organization As Well 
As Sequence 301 

Functionally Important Genome Regions 
Show Up As Islands of Conserved DNA 
Sequence 302 
































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Genome Comparisons Show That Vertébrate 
Genomes Gain and Lose DNA Rapidly 
Sequence Conservaron Allows Us to Trace 
Even the Most Distant Evolutionary 

Mobile Genetic Elements Encode the 
Components They Need for Movement 
The Human Genome Contains Two Major 
Families of Transposable Sequences 
Viruses Can Move Between Cells and Organisms 
Retroviruses Reverse the Normal Flow of 
Genetic Information 

The Nucleotide Sequences of Human Genomes 
Show How Our Genes Are Arranged 
Accelerated Changes in Conserved Genome 
Sequences Help Reveal What Makes Us 

Genome Variation Contributes to Our 
Individuality—But How? 

Differences in Gene Regulation May Help 
Explain How Animáis With Similar Genomes 
Can Be So Different 

Essential Concepts 

Chapter 10 

Modern Recombinant DNA Technology 


Restriction Nucleases Cut DNA Molecules 
at Specific Sites 

Gel Electrophoresis Separates DNA Fragments 
of Different Sizes 

Bands of DNA in a Gel Can Be Visualized Using 
Fluorescent Dyes or Radioisotopes 
Hybridization Provides a Sensitive Way to 
Detect Specific Nucleotide Sequences 

DNA Cloning Begins with Genome 
Fragmentation and Production of 
Recombinant DNAs 

Recombinant DNA Can Be Inserted Into 
Plasmid Vectors 

Recombinant DNA Can Be Copied Inside 
Bacterial Cells 

Genes Can Be Isolated from a DNA Library 
cDNA Libraries Represent the mRNAs Produced 
by Particular Cells 

PCR Uses a DNA Polymerase to Amplify 
Selected DNA Sequences in a Test Tube 

Múltiple Cycles of Amplif¡catión In Vitro 
Generate Billions of Copies of the Desired 
Nucleotide Sequence 337 

PCR ¡s Also Used for Diagnostic and Forensic 

Applications 338 


Whole Genomes Can Be Sequenced Rapidly 341 

Next-Generation Sequencing Techniques Make 

Genome Sequencing Faster and Cheaper 343 

Comparative Genome Analyses Can Identify 

Genes and Predict Their Function 346 

Analysis of mRNAs By Microarray or RNA-Seq 

Provides a Snapshot of Gene Expression 346 

In Situ Hybridization Can Reveal When and 

Where a Gene Is Expressed 347 

Repórter Genes Allow Specific Proteins to be 

Tracked in Living Cells 347 

The Study of Mutants Can Help Reveal the 

Function of a Gene 348 

RNA Interference (RNAi) Inhibits the Activity 

of Specific Genes 349 

A Known Gene Can Be Deleted or Replaced 

With an Altered Versión 350 

Mutant Organisms Provide Useful Models 

of Human Disease 352 

Transgenic Plants Are Important for Both 

Cell Biology and Agriculture 352 

Even Rare Proteins Can Be Made in Large 

Amounts Using Cloned DNA 354 

Essential Concepts 355 

Questions 356 

Chapter 11 

Membrane Structure 359 


Membrane Lipids Form Bilayers ¡n Water 361 

The Lipid Bilayer Is a Flexible Two-dimensional 

Fluid 364 

The Fluidity of a Lipid Bilayer Depends on Its 

Composition 365 

Membrane Assembly Begins ¡n the ER 366 

Certain Phospholipids Are Confined to One 

Side of the Membrane 367 


Membrane Proteins Associate with the Lipid 

Bilayer in Different Ways 370 

A Polypeptide Chain Usually Crosses the 

Lipid Bilayer as an a Helix 371 

Membrane Proteins Can Be Solubilized ¡n 

Detergents 372 

We Know the Complete Structure of 

Relatively Few Membrane Proteins 373 

The Plasma Membrane Is Reinforced by the 

Underlying Cell Cortex 374 





























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Detailed Contents 

A Cell Can Restrict the Movement of Its 
Membrane Proteins 

The Cell Surface Is Coated with Carbohydrate 

Essential Concepts 



Transport Across Cell Membranes 


Lipid Bilayers Are Impermeable to lons and 
Most Uncharged Polar Molecules 
The Ion Concentrations Inside a Cell Are Very 
Different from Those Outside 
Differences ¡n the Concentration of Inorganic 
lons Across a Cell Membrane Create a 
Membrane Potential 
Cells Contain Two Classes of Membrane 

Transport Proteins: Transporters and Channels 
Solutes Cross Membranes by Either Passive 
or Active Transport 

Both the Concentration Gradient and Membrane 
Potential Influence the Passive Transport of 
Charged Solutes 

Water Moves Passively Across Cell Membranes 
Down Its Concentration Gradient—a Process 
Called Osmosis 

Passive Transporters Move a Solute Along Its 
Electrochemical Gradient 
Pumps Actively Transport a Solute Against Its 
Electrochemical Gradient 
The Na + Pump in Animal Cells Uses Energy 
Supplied by ATPto Expel Na + and Bring 
in K + 

The Na + Pump Generates a Steep 

Concentration Gradient of Na + Across the 
Plasma Membrane 

Ca 2+ Pumps Keep the Cytosolic Ca 2+ 
Concentration Low 

Coupled Pumps Exploit Solute Gradients to 
Mediate Active Transport 
The Electrochemical Na + Gradient Drives 
Coupled Pumps in the Plasma Membrane 
of Animal Cells 

Electrochemical H + Gradients Drive Coupled 
Pumps in Plants, Fungi, and Bacteria 


Ion Channels Are lon-selective and Gated 
Membrane Potential Is Governed by the 

Permeability of a Membrane to Specific lons 
Ion Channels Randomly Snap Between Open 
and Closed States 

Different Types of Stimuli Influence the 
Opening and Closing of Ion Channels 

Voltage-gated Ion Channels Respond to the 

Membrane Potential 403 



Action Potentials Allow Rapid Long-Distance 

Communication Along Axons 404 

Action Potentials Are Mediated by Voltage- 

gated Catión Channels 405 

Voltage-gated Ca 2+ Channels in Nerve 
Termináis Convert an Electrical Signal 
into a Chemical Signal 409 

Transmitter-gated Ion Channels in the 
Postsynaptic Membrane Convert the 
Chemical Signal Back into an Electrical Signal 410 
Neurotransmitters Can Be Excitatory or 

Inhibitory 411 

Most Psychoactive Drugs Affect Synaptic 
Signaling by Binding to Neurotransmitter 
Receptors 413 

The Complexity of Synaptic Signaling Enables 

Us to Think, Act, Learn, and Remember 413 

Optogenetics Uses Light-gated Ion Channels 
to Transiently Actívate or Inactivate Neurons 
in Living Animáis 414 

Essential Concepts 415 

Questions 417 

Chapter 13 

How Cells Obtain Energy From Food 419 


Food Molecules Are Broken Down in 

Three Stages 421 

Glycolysis Extracts Energy from the Splitting 

of Sugar 422 

Glycolysis Produces Both ATP and NADH 423 

Fermentations Can Produce ATP in the 

Absence of Oxygen 425 

Glycolytic Enzymes Couple Oxidation to Energy 
Storage ¡n Activated Carriers 426 

Several Organic Molecules Are Converted 

to Acetyl CoA in the Mitochondrial Matrix 430 

The Citric Acid Cycle Generates NADH by 

Oxidizing Acetyl Groups to CO 2 430 

Many Biosynthetic Pathways Begin with 

Glycolysis or the Citric Acid Cycle 433 

Electron Transport Drives the Synthesis of the 

Majority of the ATP in Most Cells 438 


Catabolic and Anabolic Reactions Are 

Organized and Regulated 440 

Feedback Regulation Allows Cells to Switch from 
Glucose Breakdown to Glucose Synthesis 440 

Cells Store Food Molecules in Special Reservoirs 
to Prepare for Periods of Need 441 




























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Detailed Contents 

Essential Concepts 

Chapter 14 

Energy Generation in Mitochondria 
and Chloroplasts 

Cells Obtain Most of Their Energy by a 
Membrane-based Mechanism 
Chemiosmotic Coupling ¡s an Ancient Process, 
Preserved ¡n Present-Day Cells 



Mitochondria Can Change Their Shape, 

Location, and Number to Suit a Cell's Needs 
A Mitochondrion Contains an Outer Membrane, 
an Inner Membrane, and Two Internal 

The Citric Acid Cycle Generates the High-Energy 
Electrons Required for ATP Production 
The Movement of Electrons is Coupled to the 
Pumping of Protons 
Protons Are Pumped Across the Inner 

Mitochondrial Membrane by Proteins in the 
Electron-Transport Chain 
Proton Pumping Produces a Steep 

Electrochemical Proton Gradient Across the 
Inner Mitochondrial Membrane 
ATP Synthase Uses the Energy Stored in the 
Electrochemical Proton Gradient to Produce 

Coupled Transport Across the Inner 

Mitochondrial Membrane Is Also Driven by 
the Electrochemical Proton Gradient 
The Rapid Conversión of ADP to ATP in 
Mitochondria Maintains a High ATP/ADP 
Ratio in Cells 

Cell Respiration Is Amazingly Efficient 

Protons Are Readily Moved by the Transfer of 

The Redox Potential Is a Measure of Electron 

Electron Transfers Release Large Amounts 
of Energy 

Metals Tightly Bound to Proteins Form Versatile 
Electron Carriers 

Cytochrome c Oxidase Catalyzes the Reduction 
of Molecular Oxygen 

Chloroplasts Resemble Mitochondria but Have 
an Extra Compartment—the Thylakoid 
Photosynthesis Generates—Then Consumes— 

Chlorophyll Molecules Absorb the Energy of 

Excited Chlorophyll Molecules Funnel Energy 

into a Reaction Center 472 

A Pair of Photosystems Cooperate to Generate 
Both ATP and NADPH 473 

Oxygen Is Generated by a Water-Splitting 

Complex Associated with Photosystem II 474 

The Special Pair in Photosystem I Receives its 

Electrons from Photosystem II 475 

Carbón Fixation Uses ATP and NADPH to 

Convert CO 2 into Sugars 476 

Sugars Generated by Carbón Fixation Can Be 
Stored As Starch or Consumed to Produce 
ATP 478 


Oxidative Phosphorylation Evolved in Stages 479 
Photosynthetic Bacteria Made Even Fewer 

Demands on Their Environment 480 

The Lifestyle of Methanococcus Suggests That 

Chemiosmotic Coupling Is an Ancient Process 481 

Essential Concepts 482 

Questions 483 

Chapter 15 

Intracellular Compartments and 

Protein Transport 487 


Eukaryotic Cells Contain a Basic Set of 

Membrane-enclosed Organelles 488 

Membrane-enclosed Organelles Evolved in 

Different Ways 491 


Proteins Are Transported into Organelles by 

Three Mechanisms 492 

Signal Sequences Direct Proteins to the Correct 
Compartment 494 

Proteins Enter the Nucleus Through Nuclear 

Pores 495 

Proteins Unfold to Enter Mitochondria and 

Chloroplasts 497 

Proteins Enter Peroxisomes from Both the 

Cytosol and the Endoplasmic Reticulum 498 

Proteins Enter the Endoplasmic Reticulum 

While Being Synthesized 498 

Soluble Proteins Made on the ER Are Released 

into the ER Lumen 499 

Start and Stop Signáis Determine the 

Arrangement of a Transmembrane Protein 

in the Lipid Bilayer 501 


Transport Vesicles Carry Soluble Proteins and 

Membrane Between Compartments 503 

Vesicle Budding Is Driven by the Assembly of a 

Protein Coat 504 



























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Vesicle Docking Depends on Tethers and 


Most Proteins Are Covalently Modified ¡n the ER 
Exit from the ER Is Controlled to Ensure Protein 

The Size of the ER Is Controlled by the Demand 
for Protein 

Proteins Are Further Modified and Sorted in 
the Golgi Apparatus 

Secretory Proteins Are Released from the Cell 
by Exocytosis 
Specialized Phagocytic Cells Ingest Large 

Fluid and Macromolecules Are Taken Up by 

Receptor-mediated Endocytosis Provides a 
Specific Route into Animal Cells 
Endocytosed Macromolecules Are Sorted in 

Lysosomes Are the Principal Sites of 
Intracellular Digestión 

Essential Concepts 

Cell Signaling 

Signáis Can Act over a Long or Short Range 
Each Cell Responds to a Limited Set of 
Extracellular Signáis, Depending on Its 
History and Its Current State 
A Cell's Response to a Signal Can Be Fast 
or Slow 

Some Hormones Cross the Plasma Membrane 
and Bind to Intracellular Receptors 
Some Dissolved Gases Cross the Plasma 
Membrane and Actívate Intracellular 
Enzymes Directly 

Cell-Surface Receptors Relay Extracellular 
Signáis via Intracellular Signaling Pathways 
Some Intracellular Signaling Proteins Act as 
Molecular Switches 

Cell-Surface Receptors Fall into Three Main 

lon-channel-coupled Receptors Convert 
Chemical Signáis into Electrical Ones 

Stimulation of GPCRs Activates G-Protein 

Some Bacterial Toxins Cause Disease by 
Altering the Activity of G Proteins 
Some G Proteins Directly Regúlate Ion Channels 

Many G Proteins Actívate Membrane-bound 
Enzymes that Produce Small Messenger 
Molecules 543 

The Cyclic AMP Signaling Pathway Can Activate 
Enzymes and Turn On Genes 544 

The Inositol Phospholipid Pathway Triggers a 

Rise in Intracellular Ca 2+ 546 

A Ca 2+ Signal Triggers Many Biological 

Processes 548 

GPCR-Triggered Intracellular Signaling 

Cascades Can Achieve Astonishing Speed, 
Sensitivity, and Adaptability 549 


Activated RTKs Recruit a Complex of 

Intracellular Signaling Proteins 552 

Most RTKs Activate the Monomeric GTPase 

Ras 553 

RTKs Activate Pl 3-Kinase to Produce Lipid 

Docking Sites in the Plasma Membrane 555 

Some Receptors Activate a Fast Track to 

the Nucleus 558 

Cell—Cell Communication Evolved 

Independently ¡n Plants and Animáis 559 

Protein Kinase Networks Intégrate Information 

to Control Complex Cell Behaviors 560 

Essential Concepts 561 

Questions 563 

Chapter 17 

Cytoskeleton 565 


Intermedíate Filaments Are Strong and Ropelike 567 
Intermediate Filaments Strengthen Cells 

Against Mechanical Stress 569 

The Nuclear Envelope Is Supported by a 

Meshwork of Intermedíate Filaments 570 


Microtubules Are Hollow Tubes with 

Structurally Distinct Ends 572 

The Centrosome Is the Major Microtubule- 

organizing Center in Animal Cells 573 

Growing Microtubules Display Dynamic 

Instability 574 

Dynamic Instability ¡s Driven by GTP Hydrolysis 574 
Microtubule Dynamics Can be Modified by 

Drugs 575 

Microtubules Organize the Cell Interior 576 

Motor Proteins Drive Intracellular Transport 577 

Microtubules and Motor Proteins Position 

Organelles ¡n the Cytoplasm 578 

Cilia and Flagella Contain Stable Microtubules 

Moved by Dynein 579 


Actin Filaments AreThin and Flexible 584 































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Actin and Tubulin Polymerize by Similar 

Many Proteins Bind to Actin and Modify 
Its Properties 

A Cortex Rich in Actin Filaments Underlies the 
Plasma Membrane of Most Eukaryotic Cells 
Cell Crawling Depends on Cortical Actin 
Actin Associates with Myosin to Form 
Contractile Structures 

Extracellular Signáis Can Alter the Arrangement 
of Actin Filaments 

Muscle Contraction Depends on Interacting 
Filaments of Actin and Myosin 
Actin Filaments Slide Against Myosin Filaments 
During Muscle Contraction 
Muscle Contraction Is Triggered by a Sudden 
Rise in Cytosolic Ca 2+ 

Different Types of Muscle Cells Perform 
Different Functions 

Essential Concepts 


The Cell-Division Cycle 

The Eukaryotic Cell Cycle Usually Ineludes Four 

A Cell-Cycle Control System Triggers the Major 
Processes of the Cell Cycle 
Cell-Cycle Control is Similar in All Eukaryotes 
The Cell-Cycle Control System Depends on 
Cyclically Activated Protein Kinases called 

Different Cyclin-Cdk Complexes Trigger 
Different Steps in the Cell Cycle 
Cyclin Concentrations are Regulated by 
Transcription and by Proteolysis 
The Activity of Cyclin-Cdk Complexes Depends 
on Phosphorylation and Dephosphorylation 
Cdk Activity Can be Blocked by Cdk Inhibitor 

The Cell-Cycle Control System Can Pause the 
Cycle in Various Ways 


Cdks are Stably Inactivated in Gi 
Mitogens Promote the Production of the Cyclins 
that Stimulate Cell División 
DNA Damage Can Temporarily Halt Progression 
Through Gi 

Cells Can Delay División for Prolonged Periods 
by Entering Specialized Nondividing States 

S-Cdk Initiates DNA Replication and Blocks 

Re-Replication 617 

Incomplete Replication Can Arrest the Cell 

Cycle in G 2 618 

M PHASE 618 

M-Cdk Drives Entry Into M Phase and Mitosis 618 

Cohesins and Condensins Help Configure 

Duplicated Chromosomes for Separation 619 

Different Cytoskeletal Assemblies Carry 

Out Mitosis and Cytokinesis 619 

M Phase Occurs in Stages 620 


Centrosomes Duplícate To Help Form the 

Two Poles of the Mitotic Spindle 621 

The Mitotic Spindle Starts to Assemble ¡n 

Prophase 624 

Chromosomes Attach to the Mitotic Spindle 

at Prometaphase 624 

Chromosomes Assist in the Assembly of the 

Mitotic Spindle 626 

Chromosomes Line Up at the Spindle Equator 

at Metaphase 626 

Proteolysis Triggers Sister-Chromatid Separation 
at Anaphase 627 

Chromosomes Segregate During Anaphase 627 

An Unattached Chromosome Will Prevent 

Sister-Chromatid Separation 629 

The Nuclear Envelope Re-forms at Telophase 629 


The Mitotic Spindle Determines the Plañe of 

Cytoplasmic Cleavage 630 

The Contractile Ring of Animal Cells Is Made 

of Actin and Myosin Filaments 631 

Cytokinesis ¡n Plant Cells Involves the 

Formation of a New Cell Wall 632 

Membrane-Enclosed Organelles Must Be 
Distributed to Daughter Cells When a 
Cell Divides 632 

Apoptosis Helps Regúlate Animal Cell Numbers 634 
Apoptosis Is Mediated by an Intracellular 

Proteolytic Cascade 634 

The Intrinsic Apoptotic Death Program Is 
Regulated by the Bcl2 Family of Intracellular 
Proteins 636 

Extracellular Signáis Can Also Induce Apoptosis 637 
Animal Cells Require Extracellular Signáis 

to Survive, Grow, and Divide 637 

Survival Factors Suppress Apoptosis 638 

Mitogens Stimulate Cell División by Promoting 

Entry into S Phase 639 

Growth Factors Stimulate Cells to Grow 639 

Some Extracellular Signal Proteins Inhibit 

Cell Survival, División, or Growth 640 
































LibertadDigital \ 2015 

Detailed Contents 

Essential Concepts 


Sexual Reproduction and the Power 

of Genetics 


Sexual Reproduction Involves Both Diploid and 
Haploid Cells 

Sexual Reproduction Generates Genetic 

Sexual Reproduction Gives Organisms a 
Competitive Advantage in a Changing 

Meiosis Involves One Round of DNA Replication 
Followed by Two Rounds of Cell División 
Meiosis Requires the Pairing of Duplicated 
Homologous Chromosomes 
Crossing-Over Occurs Between the Duplicated 
Maternal and Paternal Chromosomes in Each 

Chromosome Pairing and Crossing-Over 

Ensure the Proper Segregation of Homologs 
The Second Meiotic División Produces Haploid 
Daughter Cells 

Haploid Gametes Contain Reassorted Genetic 

Meiosis Is Not Flawless 

Fertilization Reconstitutes a Complete Diploid 

Mendel Studied Traits That Are Inherited in 
a Discrete Fashion 

Mendel Disproved the Alternative Theories 
of Inheritance 

Mendel's Experiments Revealed the Existence 
of Dominant and Recessive Alíeles 
Each Gamete Carries a Single Alíele for Each 

Mendel's Law of Segregation Applies to All 
Sexually Reproducing Organisms 
Alíeles for Different Traits Segregate 

The Behavior of Chromosomes During Meiosis 
Underlies Mendel's Laws of Inheritance 
Even Genes on the Same Chromosome Can 
Segregate Independently by Crossing-Over 
Mutations in Genes Can Cause a Loss of 
Function or a Gain of Function 
Each of Us Carries Many Potentially Harmful 
Recessive Mutations 

The Classical Genetic Approach Begins with 
Random Mutagenesis 

Genetic Screens Identify Mutants Deficient 

in Specific Cell Processes 668 

Conditional Mutants Permit the Study of Lethal 

Mutations 670 

A Complementation Test Reveáis Whether Two 

Mutations Are in the Same Gene 671 

Rapid and Cheap DNA Sequencing Has 

Revolutionized Human Genetic Studies 672 

Linked Blocks of Polymorphisms Have Been 

Passed Down from Our Ancestors 672 

Our Genome Sequences Provide Clues to our 

Evolutionary History 673 

Polymorphisms Can Aid the Search for Mutations 
Associated with Disease 674 

Genomics Is Accelerating the Discovery of 
Rare Mutations that Predispose Us to 
Serious Disease 675 

Essential Concepts 678 

Questions 679 

Chapter 20 

Cell Communities: Tissues, Stem Cells, 

and Cáncer 683 


Plant Cells Have Tough External Walls 685 

Cellulose Microfibrils Give the Plant Cell Wall 

Its Tensile Strength 686 

Animal Connective Tissues Consist Largely of 

Extracellular Matrix 688 

Collagen Provides Tensile Strength in Animal 

Connective Tissues 688 

Cells Organize the Collagen That They Secrete 690 
Integrins Couple the Matrix Outside a Cell to 

the Cytoskeleton Inside It 691 

Gels of Polysaccharides and Proteins Fill 

Spaces and Resist Compression 692 

Epithelial Sheets Are Polarized and Rest on a 

Basal Lamina 695 

Tight Junctions Make an Epithelium Leak- 
proof and Sepárate Its Apical and Basal 
Surfaces 696 

Cytoskeleton-linked Junctions Bind Epithelial 
Cells Robustly to One Another and to the 
Basal Lamina 697 

Gap Junctions Allow Cytosolic Inorganic lons 

and Small Molecules to Pass from Cell to Cell 700 


Tissues Are Organized Mixtures of Many 

Cell Types 703 

Different Tissues Are Renewed at Different 

Rates 705 

Stem Cells Generate a Continuous Supply 

of Terminally Differentiated Cells 705 






























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Specific Signáis Maintain Stem-Cell Populations 707 
Stem Cells Can Be Used to Repair Lost or 

Damaged Tissues 708 

Therapeutic Cloning and Reproductive Cloning 

Are Very Different Enterprises 710 

Induced Pluripotent Stem Cells Provide a 

Convenient Source of Human ES-like Cells 711 


Cáncer Cells Proliferate, Invade, and Metastasize 712 
Epidemiological Studies Identify Preventable 

Causes of Cáncer 713 

Cancers Develop by an Accumulation of 

Mutations 714 

Cáncer Cells Evolve, Giving Them an 

Increasingly Competitive Advantage 715 

Two Main Classes of Genes Are Critical for 
Cáncer: Oncogenes and Tumor Suppressor 
Genes 717 

Cancer-causing Mutations Cluster in a Few 

Fundamental Pathways 719 

Colorectal Cáncer lllustrates How Loss of a 

Tumor Suppressor Gene Can Lead to Cáncer 719 

An Understanding of Cáncer Cell Biology 

Opens the Way to New Treatments 720 

Essential Concepts 724 

Questions 726 

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LibertadDigital \ 2015 

Cells: The Fundamental Units 
of Life 

What does it mean to be living? Petunias, people, and pond scum are all 
alive; stones, sand, and summer breezes are not. But what are the fun¬ 
damental properties that characterize living things and distinguish them 
from nonliving matter? 

The answer begins with a basic fact that is taken for granted now, but 
marked a revolution in thinking when flrst established 175 years ago. 
All living things (or organisms) are built from cells: small, membrane- 
enclosed units filled with a concentrated aqueous solution of Chemicals 
and endowed with the extraordinaiy ability to create copies of them- 
selves by growing and then dividing in two. The simplest forms of life are 
solitary cells. Higher organisms, including ourselves, are communities of 
cells derived by growth and división from a single founder cell. Eveiy ani¬ 
mal or plant is a vast colony of individual cells, each of which performs 
a specialized function that is regulated by intricate systems of cell-to-cell 

Cells, therefore, are the fundamental units of life. Thus it is to cell biol- 
ogy —the study of cells and their structure, function, and behavior—that 
we must look for an answer to the question of what life is and how it 
works. With a deeper understanding of cells, we can begin to tackle the 
grand historical problems of life on Earth: its mysterious origins, its stun- 
ning diversity produced by billions of years of evolution, and its invasión 
of every conceivable habitat. At the same time, cell biology can provide 
us with answers to the questions we have about ourselves: Where did we 
come from? How do we develop from a single fertilized egg cell? How is 
each of us similar to—yet different from—everyone else on Earth? Why do 
we get sick, grow oíd, and die? 






LibertadDigital \ 2015 


Cells: The Fundamental Units of Life 

In this chapter, we begin by looking at the great variety of forms that cells 
can show, and we take a preliminaiy glimpse at the Chemical machineiy 
that all cells have in common. We then consider how cells are made vis¬ 
ible under the microscope and what we see when we peer inside them. 
Finally, we discuss how we can exploit the similarities of living things to 
achieve a coherent understanding of all forms of life on Earth—frorn the 
tiniest bacterium to the mightiest oak. 


Cell biologists often speak of "the cell" without specifying any particu¬ 
lar cell. But cells are not all alike; in fact, they can be wildly different. 
Biologists estímate that there may be up to 100 million distinct species 
of living things on our planet. Before delving deeper into cell biology, we 
must take stock: What does a bacterium have in common with a butter- 
fly? What do the cells of a rose have in common with those of a dolphin? 
And in what ways do the plethora of cell types within an individual mul- 
ticellular organism differ? 

Cells Vary Enormously ¡n Appearance and Function 

Let us begin with size. A bacterial cell—say a Lactobacillus in a piece of 
cheese—is a few micrometers, or pm, in length. That's about 25 times 
smaller than the width of a human hair. A frog egg—which is also a single 
cell—has a diameter of about 1 millimeter. If we scaled them up to make 
the Lactobacillus the size of a person, the frog egg would be half a mile 

Cells vary just as widely in their shape (Figure 1 -1 ). A typical nerve cell in 
your brain, for example, is enormously extended; it sends out its electrical 
signáis along a fine protrusion that is 10,000 times longer than it is thick, 
and it receives signáis frorn other nerve cells through a mass of shorter 
processes that sprout frorn its body like the branches of a tree (see Figure 
1-1 A). A Paramecium in a drop of pond water is shaped like a submarine 
and is covered with thousands of cilia —hairlike extensions whose sinu- 
ous beating sweeps the cell forward, rotating as it goes (Figure 1-1B). 
A cell in the surface layer of a plant is squat and immobile, surrounded 

100 nm 25 nm 10 nm 5|im 1 pm 

Figure 1-1 Cells come in a variety of shapes and sizes. Note the very different scales of these micrographs. (A) Drawing of a single 
nerve cell frorn a mammalian brain. This cell has a huge branching tree of processes, through which it receives signáis from as many 
as 100,000 other nerve cells. (B) Paramecium. This protozoan—a single giant cell—swims by means of the beating cilia that cover its 
surface. (C) Chlamydomonas. This type of single-celled green algae is found all over the world—in soil, fresh water, oceans, and even 
in the snow at the top of mountains. The cell makes its food like plants do—via photosynthesis—and it pulís itself through the water 
using its paired flagella to do the breaststroke. (D) Saccharomyces cerevisiae. This yeast cell, used in baking bread, reproduces itself 
by a process called budding. (E) Helicobacter pylori. This bacterium—a causative agent of stomach ulcers—uses a handful of whiplike 
flagella to propel itself through the stomach lining. (A, copyright Herederos de Santiago Ramón y Cajal, 1899; B, courtesy of Anne 
Fleury, Michel Laurent, and André Adoutte; C, courtesy of Brian Piasecki; E, courtesy of Yutaka Tsutsumi.) 

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Unity and Diversity of Cells 

by a rigid box of cellulose with an outer waterproof coating of wax. A 
neutrophil or a macrophage in the body of an animal, by contrast, crawls 
through tissues, constantly pouring itself into new shapes, as it searches 
for and engulfs debris, foreign microorganisms, and dead or dying cells. 
And so on. 

Cells are also enormously diverse in their Chemical requirements. Some 
require oxygen to live; for others this gas is deadly. Some cells consume 
little more than air, sunlight, and water as their raw materials; others 
need a complex mixture of molecules produced by other cells. 

These differences in size, shape, and Chemical requirements often reflect 
differences in cell function. Some cells are specialized faetones for the 
production of particular substances, such as hormones, starch, fat, látex, 
or pigments. Others are engines, like muscle cells that bum fuel to do 
mechanical work. Still others are electricity generators, like the modified 
muscle cells in the electric eel. 

Some modifleations specialize a cell so much that they spoil its chances 
of leaving any descendants. Such specialization would be senseless 
for a cell that lived a solitary life. In a multicellular organism, however, 
there is a división of labor among cells, allowing some cells to become 
specialized to an extreme degree for particular tasks and leaving them 
dependent on their fellow cells for many basic requirements. Even the 
most basic need of all, that of passing on the genetic instructions of the 
organism to the next generation, is delegated to specialists—the egg and 
the sperm. 

Living Cells All Have a Similar Basic Chemistry 

Despite the extraordinary diversity of plants and animáis, people have 
recognized from time immemorial that these organisms have something 
in common, something that entitles them all to be called living things. 
But while it seemed easy enough to recognize life, it was remarkably dif- 
ficult to say in what sense all living things were alike. Textbooks had to 
settle for defining life in abstract general terms related to growth, repro- 
duction, and an ability to respond to the environment. 

The discoveries of biochemists and molecular biologists have provided 
an elegant solution to this awkward situation. Although the cells of all 
living things are infinitely varied when viewed from the outside, they 
are fundamentally similar inside. We now know that cells resemble one 
another to an astonishing degree in the details of their chemistry. They are 
composed of the same sorts of molecules, which particípate in the same 
types of Chemical reactions (discussed in Chapter 2). In all organisms, 
genetic information—in the form of genes —is carried in DNA molecules. 
This information is written in the same Chemical code, constructed out 
of the same Chemical building blocks, interpreted by essentially the same 
Chemical machinery, and replicated in the same way when an organism 
reproduces. Thus, in every cell, the long DNA polymer chains are made 
from the same set of four monomers, called nucleotides, strung together 
in different sequences like the letters of an alphabet to convey informa¬ 
tion. In every cell, the information encoded in the DNA is read out, or 
transcribed, into a chemically related set of polymers called RNA. A sub¬ 
set of these RNA molecules is in turn translated into yet another type of 
polymer called a protein. This flow of information—from DNA to RNA 
to protein—is so fundamental to life that it is referred to as the central 
dogma (Figure 1-2). 

The appearance and behavior of a cell are dictated largely by its 
protein molecules, which serve as structural supports, Chemical catalysts, 


"Life" is easy to recognize but 
difficult to define. According to one 
popular biology text, living things: 

1. Are highly organized compared 
to natural inanimate objeets. 

2. Display homeostasis, maintaining 
a relatively constant internal 

3. Reproduce themselves. 

4. Grow and develop from simple 

5. Take energy and matter from the 
environment and transform it. 

6. Respond to stimuli. 

7. Show adaptation to their 

Score a person, a vacuum cleaner, 
and a potato with respect to these 

DNA synthesis 


!■ 11■ ■ I I M II II 

amino acids 

Figure 1-2 In all living cells, genetic 
information flows from DNA to RNA 
(transcription) and from RNA to protein 
(translation)—a sequence known as 
the central dogma. The sequence of 
nucleotides in a particular segment of 
DNA (a gene) is transcribed into an RNA 
molecule, which can then be translated into 
the linear sequence of amino acids of a 
protein. Only a small part of the gene, RNA, 
and protein are shown. 

LibertadDigital \ 2015 

CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-3 All living organisms are 
constructed from cells. A colony 
of bacteria, a butterfly, a rose, and a 
dolphin are all made of cells that have a 
fundamentally similar chemistry and opérate 
according to the same basic principies. 

(A, courtesy of Janice Carr; C, courtesy of 
the John Innes Foundation; D, courtesy of 
Jonathan Gordon, IFAW.) 

molecular motors, and so on. Proteins are built from amino acids, and all 
organisms use the same set of 20 amino acids to make their proteins. 
But the amino acids are linked in different sequences, giving each type 
of protein molecule a different three-dimensional shape, or conforma- 
tion, just as different sequences of letters spell different words. In this 
way, the same basic biochemical machinery has served to generate the 
whole gamut of life on Earth (Figure 1-3). A more detailed discussion 
of the structure and function of proteins, RNA, and DNA is presented in 
Chapters 4 through 8. 

If cells are the fundamental unit of living matter, then nothing less than 
a cell can truly be called living. Viruses, for example, are compact pack- 
ages of genetic information—in the form of DNA or RNA—encased in 
protein but they have no ability to reproduce themselves by their own 
efforts. Instead, they get themselves copied by parasitizing the reproduc- 
tive machinery of the cells that they invade. Thus, viruses are Chemical 
zombies: they are inert and inactive outside their host cells, but they can 
exert a malign control over a cell once they gain entry. 


Mutations are mistakes in the DNA 
that change the genetic plan from 
the previous generation. Imagine 
a shoe factory. Would you expect 
mistakes (i.e., unintentional changes) 
in copying the shoe design to lead 
to improvements in the shoes 
produced? Explain your answer. 

All Present-Day Cells Have Apparently Evolved from the 
Same Ancestral Cell 

A cell reproduces by replicating its DNA and then dividing in two, passing 
a copy of the genetic instructions encoded in its DNA to each of its daugh- 
ter cells. That is why daughter cells resemble the parent cell. However, 
the copying is not always perfect, and the instructions are occasionally 
corrupted by mutations that change the DNA. For this reason, daughter 
cells do not always match the parent cell exactly. 

Mutations can create offspring that are changed for the worse (in that 
they are less able to survive and reproduce), changed for the better (in 
that they are better able to survive and reproduce), or changed in a neutral 
way (in that they are genetically different but equally viable). The struggle 
for survival eliminates the flrst, favors the second, and tolerates the third. 
The genes of the next generation will be the genes of the survivors. 

On occasion, the pattern of descent may be complicated by sexual repro- 
duction, in which two cells of the same species fuse, pooling their DNA. 
The genetic cards are then shuffled, re-dealt, and distributed in new com- 
binations to the next generation, to be tested again for their ability to 
promote survival and reproduction. 

These simple principies of genetic change and selection, applied repeat- 
edly over billions of cell generations, are the basis of evolution— the 
process by which living species become gradually modified and adapted 
to their environment in more and more sophisticated ways. Evolution 
offers a startling but compelling explanation of why present-day cells 
are so similar in their fundamentáis: they have all inherited their genetic 
instructions from the same common ancestor. It is estimated that this 
ancestral cell existed between 3.5 and 3.8 billion years ago, and we musí 

LibertadDigital \ 2015 

Cells Underthe Microscope 

suppose that it contained a prototype of the universal machinery of all 
life on Earth today. Through a very long process of mutation and natural 
selection, the descendants of this ancestral cell have gradually diverged 
to fill eveiy habitat on Earth with organisms that exploit the potential of 
the machinery in an endless variety of ways. 

Genes Provide the Instructions for Cell Form, Function, 
and Complex Behavior 

A cell's genome—that is, the entire sequence of nucleotides in an organ¬ 
ismo DNA—provides a genetic program that instructs the cell how to 
behave. For the cells of plant and animal embryos, the genome directs 
the growth and development of an adult organism with hundreds of dif- 
ferent cell types. Within an individual plant or animal, these cells can be 
extraordinarily varied, as we discuss in Chapter 20. Fat cells, skin cells, 
bone cells, and nerve cells seem as dissimilar as any cells could be. Yet 
all these differentiated cell types are generated during embryonic develop¬ 
ment from a single fertilized egg cell, and all contain identical copies of 
the DNA of the species. Their varied characters stem from the way that 
individual cells use their genetic instructions. Different cells express dif- 
ferent genes: that is, they use their genes to produce some proteins and 
not others, depending on their internal State and on cues that they and 
their ancestor cells have received from their surroundings—mainly sig¬ 
náis from other cells in the organism. 

The DNA, therefore, is not just a shopping list specifying the molecules 
that every cell musí make, and a cell is not just an assembly of all the 
ítems on the list. Each cell is capable of carrying out a variety of biologi- 
cal tasks, depending on its environment and its history, and it selectively 
uses the information encoded in its DNA to guide its activities. Later in 
this book, we will see in detail how DNA defines both the parts list of the 
cell and the rules that decide when and where these parts are to be made. 


Today, we have the technology to decipher the underlying principies 
that govern the structure and activity of the cell. But cell biology started 
without these tools. The earliest cell biologists began by simply looking 
at tissues and cells, and later breaking them open or slicing them up, 
attempting to view their contents. What they saw was to them profoundly 
baffiing—a collection of tiny and scarcely visible objects whose relation- 
ship to the properties of living matter seemed an impenetrable mysteiy. 
Nevertheless, this type of visual investigation was the first step toward 
understanding cells, and it remains essential in the study of cell biology. 
Cells were not made visible until the seventeenth century, when the 
microscope was invented. For hundreds of years afterward, all that 
was known about cells was discovered using this instrument. Light 
microscopes use visible light to illuminate specimens, and they allowed 
biologists to see for the first time the intricate structure that underpins all 
living things. 

Although these instruments now incorpórate many sophisticated 
improvements, the properties of light itself set a limit to the fineness of 
detail they reveal. Electron microscopes, invented in the 1930s, go beyond 
this limit by using beams of electrons instead of beams of light as the 
source of illumination, greatly extending our ability to see the fine details 
of cells and even making some of the larger molecules visible individ- 
ually. These and other forms of microscopy remain vital tools in the 
modern cell biology laboratory, where they continué to reveal new and 
sometimes surprising details about the way cells are built and how they 

LibertadDigital \ 2015 

CHAPTER 1 Cells: The Fundamental Units of Life 

The Invention of the Light Microscope Led to the 
Discovery of Cells 

The development of the light microscope depended on advances in the 
production of glass lenses. By the seventeenth century, lenses were pow- 
erful enough to make out details invisible to the naked eye. Using an 
instrument equipped with such a lens, Robert Hooke examined a piece 
of cork and in 1665 reported to the Royal Society of London that the cork 
was composed of a mass of minute chambers. He called these cham- 
bers "cells," based on their resemblance to the simple rooms occupied 
by monks in a monastery. The ñame stuck, even though the structures 
Hooke described were actually the cell walls that remained after the living 
plant cells inside them had died. Later, Hooke and his Dutch contempo- 
rary Antoni van Leeuwenhoek were able to observe living cells, seeing 
for the first time a world teeming with motile microscopic organisms. 

For almost 200 years, such instruments—the first light microscopes— 
remained exotic devices, available only to a few wealthy individuáis. It 
was not until the nineteenth century that microscopes began to be widely 
used to look at cells. The emergence of cell biology as a distinct Science 
was a gradual process to which many individuáis contributed, but its offi- 
cial birth is generally said to have been signaled by two publications: one 
by the botanist Matthias Schleiden in 1838 and the other by the zoolo- 
gist Theodor Schwann in 1839. In these papers, Schleiden and Schwann 
documented the results of a systematic investigation of plant and animal 
tissues with the light microscope, showing that cells were the universal 
building blocks of all living tissues. Their work, and that of other nine- 
teenth-century microscopists, slowly led to the realization that all living 
cells are formed by the growth and división of existing cells—a principie 
sometimes referred to as the cell theoiy (Figure 1-4). The implication that 

Figure 1-4 New cells form by growth and división of existing cells. (A) In 1880, Eduard Strasburger drew a living plant cell 

(a hair cell from a Tradescantia flower), which he observed dividing into two daughter cells over a period of 2.5 hours. (B) A comparable 

living plant cell photographed recently through a modern light microscope. (B, courtesy of Peter Hepler.) 

LibertadDigital \ 2015 

Cells Underthe Microscope 

living organisms do not arise spontaneously but can be generated only 
from existing organisms was hotly contested, but it was finally confirmed 
in the 1860s by an elegant set of experiments performed by Louis Pasteur. 
The principie that cells are generated only from preexisting cells and 
inherit their characteristics from them underlies all of biology and gives 
the subject a unique flavor: in biology, questions about the present are 
inescapably linked to questions about the past. To understand why 
present-day cells and organisms behave as they do, we need to under¬ 
stand their history, all the way back to the misty origins of the first cells 
on Earth. Charles Darwin provided the key insight that makes this his¬ 
tory comprehensible. His theoiy of evolution, published in 1859, explains 
how random variation and natural selection gave rise to diversity among 
organisms that share a common ancestry. When combined with the cell 
theory, the theory of evolution leads us to view all life, from its beginnings 
to the present day, as one vast family tree of individual cells. Although 
this book is primarily about how cells work today, we will encounter the 
theme of evolution again and again. 

Light Microscopes Allow Examination of Cells and Some of 
Their Components 

If you cut a veiy thin slice from a suitable plant or animal tissue and view 
it using a light microscope, you will see that the tissue is divided into 
thousands of small cells. These may be either closely packed or separated 
from one another by an extracellular matrix, a dense material often made 
of protein fibers embedded in a polysaccharide gel (Figure 1-5). Each cell 
is typically about 5-20 |im in diameter. If you have taken care of your 
specimen so that its cells remain alive, you will be able to see partióles 
moving around inside individual cells. And if you watch patiently, you 
may even see a cell slowly change shape and divide into two (see Figure 
1-4 and a speeded-up video of cell división in a frog embryo in Movie 1.1). 
To see the intemal structure of a cell is difficult, not only because the 
parts are small, but also because they are transparent and mostly color- 
less. One way around the problem is to stain cells with dyes that color 
particular components differently (see Figure 1-5). Alternatively, one can 
exploit the fact that cell components differ slightly from one another in 


You have embarked on an ambitious 
research project: to create life in a 
test tube. You boil up a rich mixture 
of yeast extract and amino acids 
in a flask along with a sprinkling 
of the inorganic salts known to be 
essential for life. You seal the flask 
and allow it to cool. After several 
months, the liquid is as clear as 
ever, and there are no signs of life. 
Afriend suggests that excluding 
the air was a mistake, since most 
life as we know it requires oxygen. 
You repeat the experiment, but this 
time you leave the flask open to the 
atmosphere. To your great delight, 
the liquid becomes cloudy after a 
few days and under the microscope 
you see beautiful small cells that 
are clearly growing and dividing. 
Does this experiment prove that 
you managed to generate a novel 
life-form? How might you redesign 
your experiment to allow air 
into the flask, yet elimínate the 
possibility that contamination is 
the explanation for the results? 

(For a ready-made answer, look up 
the classic experiments of Louis 

Figure 1-5 Cells form tissues in plants 
and animáis. (A) Cells in the root tip of a 
fern. The nuclei are stained red, and each 
cell is surrounded by a thin cell wall (light 
blue). (B) Cells in the urine-collecting ducts 
of the kidney. Each duct appears in this 
cross section as a ring of closely packed 
cells (with nuclei stained red). The ring is 
surrounded by extracellular matrix, stained 
purple. (A, courtesy of James Mauseth; 

B, from P.R. Wheater et al., Functional 
Histology, 2nd ed. Edinburgh: Churchill 
Livingstone, 1987. With permission from 

LibertadDigital \ 2015 

CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-6 Some of the ¡nternal 
structures of a living cell can be seen 
with a light microscope. (A) A cell taken 
from human skin and grown ¡n culture was 
photographed through a light microscope 
using interference-contrast optics (see Panel 
1-1, pp. 10-11). The nucleus ¡s especially 
prominent. (B) A pigment cell from a frog, 
stained with fluorescent dyes and viewed 
with a confocal fluorescence microscope 
(see Panel 1-1). The nucleus is shown in 
purple, the pigment granules in red, and 
the microtubules—a class of filaments built 
from protein molecules in the cytoplasm—in 
green. (A, courtesy of Casey Cunningham; 

B, courtesy of Stephen Rogers and the 
Imaging Technology Group of the Beckman 
Institute, University of Illinois, Urbana.) 

refractive Índex, just as glass differs in refractive índex from water, caus- 
ing light rays to be deflected as they pass from the one médium into the 
other. The small differences in refractive Índex can be made visible by 
specialized optical techniques, and the resulting images can be enhanced 
further by electronic processing. 

The cell thus revealed has a distinct anatomy (Figure 1-6A). It has 
a sharply defined boundary, indicating the presence of an enclosing 
membrane. A large, round structure, the nucleus, is prominent in the 
middle of the cell. Around the nucleus and filling the cell's interior is the 
cytoplasm, a transparent substance crammed with what seems at first to 
be a jumble of miscellaneous objects. With a good light microscope, one 
can begin to distinguish and classify some of the specific components in 
the cytoplasm, but structures smaller than about 0.2 ¡im—about half the 
wavelength of visible light—cannot normally be resolved; points closer 
than this are not distinguishable and appear as a single blur. 

In recent years, however, new types of fluorescence microscopes have 
been developed that use sophisticated methods of illumination and elec¬ 
tronic image processing to see fluorescently labeled cell components in 
much finer detail (Figure 1-6B). The most recent super-resolution flu¬ 
orescence microscopes, for example, can push the limits of resolution 
down even further, to about 20 nanometers (nm). That is the size of a 
single ribosome, a large macromolecular complex composed of 80-90 
individual proteins and RNA molecules. 

The Fine Structure of a Cell Is Revealed by Electron 

For the highest magnification and best resolution, one must turn to an 
electrón microscope, which can reveal details down to a few nano¬ 
meters. Cell samples for the electrón microscope require painstaking 
preparation. Even for light microscopy, a tissue often has to be jrxed (that 
is, preserved by pickling in a reactive Chemical solution), supported by 
embedding in a solid wax or resin, cut or sectioned into thin slices, and 
stained before it is viewed. For electrón microscopy, similar procedures 
are required, but the sections have to be much thinner and there is no 
possibility of looking at living, wet cells. 

LibertadDigital \ 2015 

Cells Underthe Microscope 

When thin sections are cut, stained, and placed in the electrón microscope, 
much of the jumble of cell components becomes sharply resolved into 
distinct organdíes—sepárate, recognizable substructures with special- 
ized functions that are often only hazily deflned with a light microscope. 
A delicate membrane, only about 5 nm thick, is visible enclosing the cell, 
and similar membranes form the boundary of many of the organdíes 
inside (Figure 1-7A, B). The membrane that separates the interior of the 
cell frorn its external environment is called the plasma membrane, while 
the membranes surrounding organdíes are called intemal membranes. 
All of these membranes are only two molecules thick (as discussed in 
Chapter 11). With an electrón microscope, even individual large mole¬ 
cules can be seen (Figure 1-7C). 

The type of electrón microscope used to look at thin sections of tissue is 
known as a transmission electrón microscope. This is, in principie, simi¬ 
lar to a light microscope, except that it transmits a beam of electrons 
rather than a beam of light through the sample. Another type of electrón 
microscope—the scanning electrón microscope— scatters electrons off the 
surface of the sample and so is used to look at the surface detail of cells 
and other structures. A survey of the principal types of microscopy used 
to examine cells is given in Panel 1-1 (pp. 10-11). 

Figure 1-7 The fine structure of a cell 
can be seen in a transmission electrón 
microscope. (A) Thin section of a liver cell 
showing the enormous amount of detail that 
is visible. Some of the components to be 
discussed later in the chapter are labeled; 
they are identifiable by their size and shape. 
(B) Asmall región of the cytoplasm at higher 
magnification. The smallest structures that 
are clearly visible are the ribosomes, each 
of which is made of 80-90 or so individual 
large molecules. (C) Portion of a long, 
threadlike DNA molecule ¡solated from a 
cell and viewed by electrón microscopy. 

(A and B, courtesy of Daniel S. Friend; 

C, courtesy of Mei Lie Wong.) 

PANEL 1-1 



The light microscope allows us to 
magnify cells up to 1000 times and to 
resolve details as small as 0.2 pm 
(a limitation imposed by the wavelike 
nature of light, not by the quality of 
the lenses). Three things are required 
for viewing cells in a light microscope. 
First, a bright light must be focused 
onto the specimen by lenses in the 
condensen Second, the specimen must 
be carefully prepared to allow light to 
passthrough it. Third, an appropriate 
set of lenses (objective and eyepiece) 
must be arranged to focus an image of 
the specimen in the eye. 

specimen — 

the light path in e 
light microscope 



Fluorescent dyes used for staining cells are detected with the 
aid of a fluorescence microscope. This is similar to an 
ordinary light microscope except that the ¡lluminating light 
is passed through two sets of filters. The first ( fe) filters the 
light before it reaches the specimen, passing only those 
wavelengths that excite the particular fluorescent dye. The 
second (|p) blocks out this light and passes only those 
wavelengths emitted when the dye fluoresces. Dyed objects 
show up in bright color on a dark background. 


The same unstained, living 
animal cell (fibroblast) in 
culture viewed with 

(A) straightforward 
(bright-field) optics; 

(B) phase-contrast optics; 

(C) interference-contrast 
optics. The two latter 
Systems exploit differences 
in the way light travels 
through regions of the cell 
with differing refractive 
indexes. All three images 
can be obtained on the 
same microscope simply by 
¡nterchanging optical 


Most tissues are neither small enough ñor 
transparent enough to examine directly in 
the microscope. Typically, therefore, they 
e chemically fixed and cut into very thin 
slices, or sections, that can be mounted on 
a glass microscope slide and subsequently 
stained to reveal different components of 
the cells. A stained section of a plant root 
tip is shown here (D). (Courtesy of 
Catherine Kidner.) 


Dividing nudei in a fly embryo seen with a fluorescence 
microscope after staining with specific fluorescent dyes. 

Fluorescent dyes absorb light at one wavelength and 
emit it at another, longer wavelength. Some such 
dyes bind specifically to particular molecules in cells 
and can reveal their location when examined with a 
fluorescence microscope. An example is the stain for 
DNA shown here ( green ). Other dyes can be coupled 
to antibody molecules, which then serve as highly 
specific and versatile staining reagents that bind 
selectively to particular large molecules, allowing us 
to see their distribution in the cell. In the example 
shown, a microtubule protein in the mitotic spindle 
is stained red with a fluorescent antibody. (Courtesy 
of William Sullivan.) 

LibertadDigital \. 

Cells Underthe Microscope 



A confocal microscope is a specialized type of fluorescence microscope that builds up an 
image by scanning the specimen with a láser beam. The beam is focused onto a single 
point at a specific depth in the specimen, and a pinhole aperture in the detector allows 
only fluorescence emitted from this same point to be induded in the image. Scanning the 
beam across the specimen generates a sharp image of the plañe of focus—an optical 
section. A series of optical sections at different depths allows a three-dimensional image 
to be constructed. An intact insect embryo is shown here stained with a fluorescent probe 
for actin filaments. (A) Conventional fluorescence microscopy gives a blurry image due to 
the presence of fluorescent structures above and below the plañe of focus. (B) Confocal 
microscopy provides an optical section showing the individual cells dearly. (Courtesy of 
Richard Warn and Peter Shaw.) 




The electrón micrograph below 
shows a small región of a cell in 
a piece of testis. The tissue has 
been chemically fixed, 
embedded in plástic, and cut 
into very thin sections that have 
then been stained with salts of 
uranium and lead. (Courtesy of 
Daniel S. Friend.) 

The transmission electrón microscope (TEM) is in principie similar 
to a light microscope, but it uses a beam of electrons instead of a 
beam of light, and magnetic coils to focus the beam instead of 
glass lenses. The specimen, which is placed in a vacuum, must be 
very thin. Contrast is usually introduced by staining the specimen 
with electrón-dense heavy metáis that locally absorb or scatter 
electrons, removing them from the beam as it passes through 
the specimen. The TEM has a useful magnification of up to a 
| million-fold and can resolve details as small as about 1 nm in 
biological specimens. 


In the scanning electrón microscope (SEM), the specimen, which 
has been coated with a very thin film of a heavy metal, is scanned 
by a beam of electrons brought to a focus on the specimen by 
magnetic coils that act as lenses. The quantity of electrons 
scattered or emitted as the beam bombards each successive point 
on the surface of the specimen is measured by the detector, and is 
used to control the intensity of successive points in an image built 
up on a video screen. The microscope creates striking images of 
three-dimensional objects with great depth of focus and can 
resolve details down to somewhere between 3 nm and 20 nm, 
depending on the ¡nstrument. 


CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-8 How big is a cell and its components? (A) The sizes of cells and of their component parís, plus the 
units ¡n which they are measured. (B) Drawings to convey a sense of scale between living cells and atoms. Each 
panel shows an image that is magnified by a factor of 10 compared to its predecessor—producing an imaginary 
progression from a thumb, to skin, to skin cells, to a mitochondrion, to a ribosome, and ultimately to a cluster of 
atoms forming parí of one of the many protein molecules ¡n our bodies. Note that ribosomes are present inside 
mitochondria (as shown here), as well as ¡n the cytoplasm. Details of molecular structure, as shown in the last two 
panels, are beyond the power of the electrón microscope. 

Even the most powerful electrón microscopes, however, cannot visualize 
the individual atoms that make up biological molecules (Figure 1-8). To 
study the cell's key components in atomic detail, biologists have developed 
even more sophisticated tools. A technique called X-ray ciystallography, 
for example, is used to determine the precise three-dimensional structure 
of protein molecules (discussed in Chapter 4). 


Of all the types of cells revealed by the microscope, bacteria have the sim- 
plest structure and come closest to showing us life stripped down to its 
essentials. Indeed, a bacterium contains essentially no organdíes—not 
even a nucleus to hold its DNA. This property—the presence or absence 
of a nucleus—is used as the basis for a simple but fundamental classifica- 
tion of all living things. Organisms whose cells have a nucleus are called 
eukaryotes (from the Greek words cu, meaning "well" or "truly," and 
kaiyon, a "kernel" or "nucleus"). Organisms whose cells do not have a 
nucleus are called prokaiyotes (from pro, meaning "before"). The terms 

LibertadDigital \ 2015 

The Prokaryotic Cell 


spherical cells, rod-shaped cells, 

e.g., Streptococcus e.g., Escheríchia coli, 


spiral cells, 

e.g., Treponema pallidum 

"bacterium" and "prokaiyote" are often used interchangeably, although 
we will see that the categoiy of prokaryotes also ineludes another class 
of cells, the archaea (singular archaeon), which are so remotely related to 
bacteria that they are given a sepárate ñame. 

Prokaryotes are typically spherical, rodlike, or corkscrew-shaped (Figure 
1-9). They are also small—generally just a few micrometers long, 
although there are some giant species as much as 100 times longer than 
this. Prokaryotes often have a tough protective coat, or cell wall, sur- 
rounding the plasma membrane, which endoses a single compartment 
containing the cytoplasm and the DNA. In the electrón microscope, the 
cell interior typically appears as a matrix of varying texture, without any 
obvious organized intemal structure (Figure 1-10). The cells reproduce 
quickly by dividing in two. Under optimum conditions, when food is plen- 
tiful, many prokaryotic cells can duplícate themselves in as little as 20 
minutes. In 11 hours, by repeated divisions, a single prokaryote can give 
rise to more than 8 billion progeny (which exceeds the total number of 
humans presently on Earth). Thanks to their large numbers, rapid growth 
rates, and ability to exchange bits of genetic material by a process akin 
to sex, populations of prokaryotic cells can evolve fast, rapidly acquir- 
ing the ability to use a new food source or to resist being killed by a new 
antibio tic. 

Prokaryotes Are the Most Diverse and Numerous Cells 
on Earth 

Most prokaryotes live as single-celled organisms, although some join 
together to form chains, clusters, or other organized multicellular struc- 
tures. In shape and structure, prokaryotes may seem simple and limited, 
but in terms of chemistry, they are the most diverse and inventive class of 
cells. Members of this class exploit an enormous range of habitats, frorn 
hot puddles of volcanic mud to the interiors of other living cells, and they 
vastly outnumber all eukaryotic organisms on Earth. Some are aerobic, 
using oxygen to oxidize food molecules; some are strictly anaerobic and 
are killed by the slightest exposure to oxygen. As we discuss later in this 

Figure 1-9 Bacteria come in different 
shapes and sizes. Typical spherical, rodlike, 
and spiral-shaped bacteria are drawn 
to scale. The spiral cells shown are the 
organisms that cause syphilis. 


A bacterium weighs about 10 -12 g 
and can divide every 20 minutes. 

If a single bacterial cell carried on 
dividing at this rate, how long would 
it take before the mass of bacteria 
would equal that of the Earth 
(6 x 10 24 kg)? Contrast your result 
with the fact that bacteria originated 
at least 3.5 billion years ago and 
have been dividing ever since. 
Explain the apparent paradox. (The 
number of cells N in a culture at 
time t is described by the equation 
N = Nq x 2 t/G , where No is the 
number of cells at zero time and G is 
the population doubling time.) 

Figure 1-10 The bacterium Escheríchia 
coli (E. coli) has served as an important 
model organism. An electrón mlcrograph 
of a longitudinal section is shown here; 
the cell's DNA is concentrated ¡n the 
lightly stained reglón. (Courtesy of 
E. Kellenberger.) 

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Cells: The Fundamental Units of Life 

Figure 1-11 Some bacteria are photosynthetic. (A) Anabaena cylindrica forms 
long, multicellular filaments. This light micrograph shows specialized cells that 
either fix nitrogen (that is, capture N2 from the atmosphere and incorpórate ¡t into 
organic compounds; labeled H), fix C02through photosynthesis (labeled V), or 
become resistant spores (labeled S). (B) An electrón micrograph of a related species, 
Phormidium laminosum, shows the intracellular membranes where photosynthesis 
occurs. These micrographs ¡Ilústrate that even some prokaryotes can form simple 
multicellular organisms. (A, courtesy of David Adams; B, courtesy of D.P. Hill and 
C.J. Howe.) 

chapter, mitochondria —the organelles that generate energy in eukaryo- 
tic cells—are thought to have evolved from aerobic bacteria that took 
to living inside the anaerobic ancestors of today’s eukaryotic cells. Thus 
our own oxygen-based metabolism can be regarded as a product of the 
activities of bacterial cells. 

Virtually any organic, carbon-containing material—from wood to Petro¬ 
leum—can be used as food by one sort of bacterium or another. Even 
more remarkably, some prokaryotes can live entirely on inorganic sub- 
stances: they can get their carbón from CO 2 in the atmosphere, their 
nitrogen from atmospheric N 2 , and their oxygen, hydrogen, sulfur, and 
phosphorus from air, water, and inorganic minerals. Some of these 
prokaryotic cells, like plant cells, perform photosynthesis, using energy 
from sunlight to produce organic molecules from CO 2 (Figure 1-11); oth- 
ers derive energy from the Chemical reactivity of inorganic substances in 
the environment (Figure 1-12). In either case, such prokaryotes play a 
unique and fundamental part in the economy of life on Earth: other living 
things depend on the organic compounds that these cells generate from 
inorganic materials. 

Plants, too, can capture energy from sunlight and carbón from atmos¬ 
pheric CO 2 . But plants unaided by bacteria cannot capture N 2 from the 
atmosphere, and in a sense even plants depend on bacteria for photo¬ 
synthesis. It is almost certain that the organelles in the plant cell that 

Figure 1-12 A sulfur bacterium gets its energy from H2S. 

Beggiatoa, a prokaryote that lives in sulfurous environments, oxidizes 
H2S to produce sulfur and can fix carbón even in the dark. In this light 
micrograph, yellow deposits of sulfur can be seen inside both of the 
cells. (Courtesy of Ralph W. Wolfe.) 

LibertadDigital \ 2015 

perform photosynthesis—the chloroplasts—have evolved from photosyn- 
thetic bacteria that long ago found a home inside the cytoplasm of a plant 
cell ancestor. 

The World of Prokaryotes Is Divided ¡nto Two Domains: 
Bacteria and Archaea 

Traditionally, all prokaryotes have been classified together in one large 
group. But molecular studies reveal that there is a gulf within the class 
of prokaryotes, dividing it into two distinct domains called the bacteria 
and the archaea. Remarkably, at a molecular level, the members of these 
two domains differ as much from one another as either does from the 
eukaryotes. Most of the prokaryotes familiar from everyday life—the spe- 
cies that live in the soil or make us ill—are bacteria. Archaea are found 
not only in these habitats, but also in environments that are too hostile 
for most other cells: concentrated brine, the hot acid of volcanic springs, 
the airless depths of marine sediments, the sludge of sewage treatment 
plants, pools beneath the frozen surface of Antárctica, and in the acidic, 
oxygen-free environment of a cow’s stomach where they break down cel- 
lulose and generate methane gas. Many of these extreme environments 
resemble the harsh conditions that must have existed on the primitive 
Earth, where living things first evolved before the atmosphere became 
rich in oxygen. 


Eukaryotic cells, in general, are bigger and more elabórate than bacte¬ 
ria and archaea. Some live independent lives as single-celled organisms, 
such as amoebae and yeasts (Figure 1-13); others live in multicellular 
assemblies. All of the more complex multicellular organisms—including 
plants, animáis, and fungi—are formed from eukaryotic cells. 

By definition, all eukaryotic cells have a nucleus. But possession of 
a nucleus goes hand-in-hand with possession of a variety of other 
organelles, most of which are membrane-enclosed and common to 
all eukaryotic organisms. In this section, we take a look at the main 
organelles found in eukaryotic cells from the point of view of their func- 
tions, and we consider how they carne to serve the roles they have in the 
life of the eukaryotic cell. 

The Nucleus Is the Information Store of the Cell 

The nucleus is usually the most prominent organelle in a eukaryotic cell 
(Figure 1-14). It is enclosed within two concentric membranes that form 
the nuclear envelope, and it contains molecules of DNA—extremely long 
polymers that encode the genetic information of the organism. In the 
light microscope, these giant DNA molecules become visible as individual 
chromosomes when they become more compact before a cell divides 
into two daughter cells (Figure 1-15). DNA also carries the genetic infor¬ 
mation in prokaryotic cells; these cells lack a distinct nucleus not because 
they lack DNA, but because they do not keep their DNA inside a nuclear 
envelope, segregated from the rest of the cell contents. 

Figure 1-13 Yeasts are simple free-living eukaryotes. The 

cells shown in this micrograph belong to the specles of yeast, 
Saccharomyces cerevisiae, used to make dough rise and turn 
malted barley juice ¡nto beer. As can be seen in this ¡mage, the cells 
reproduce by growlng a bud and then dividing asymmetrically ¡nto a 
large mother cell and a small daughter cell; for this reason, they are 
called budding yeast. 

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CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-14 The nucleus contains most 
of the DNA in a eukaryotic cell. (A) This 
drawing of a typical animal cell shows its 
extensive system of membrane-enclosed 
organelles. The nucleus is colored brown, 
the nuclear envelope is green, and the 
cytoplasm (the interior of the cell outside 
the nucleus) is wh/te. (B) An electrón 
micrograph of the nucleus in a mammalian 
cell. Individual chromosomes are not visible 
because at this stage of the cell's growth 
its DNA molecules are dispersed as fine 
threads throughout the nucleus. (B, courtesy 
of Daniel S. Friend.) 

Mitochondria Generate Usable Energy from Food to 
Power the Cell 

Mitochondria are present in essentially all eukaryotic cells, and they are 
among the most conspicuous organelles in the cytoplasm (see Figure 
1-7B). In a fluorescence microscope, they appear as worm-shaped struc- 
tures that often form branching networks (Figure 1-16). When seen 
with an electrón microscope, individual mitochondria are found to be 
enclosed in two sepárate membranes, with the inner membrane formed 
into folds that project into the interior of the organelle (Figure 1-17). 
Microscopic examination by itself, however, gives little indication of 
what mitochondria do. Their function was discovered by breaking open 
cells and then spinning the soup of cell fragments in a centrifuge; this 

Figure 1-15 Chromosomes become 
visible when a cell is about to divide. 

As a eukaryotic cell prepares to divide, its 
DNA molecules become progressively more 
compacted (condensed), forming wormlike 
chromosomes that can be distinguished 
in the light microscope. The photographs 
show three successive steps in this process 
in a cultured cell from a newt's lung; note 
that in the last micrograph on the right, 
the nuclear envelope has broken down. 
(Courtesy of Conly L. Rieder.) 

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The Eukaryotic Cell 


Figure 1-16 Mitochondria can be variable in shape and size. This 
budding yeast cell, which contalns a green fluorescent proteln in its 
mitochondria, was viewed in a super-resolution confocal fluorescence 
microscope. In this three-dimensional image, the mitochondria are 
seen to form complex branched networks. (From A. Egner et al., Proc. 
Nati Acad. Sci. USA 99:3370-3375, 2002. With permission from the 
National Academy of Sciences.) 

separates the organdíes according to their size and density. Purified 
mitochondria were then tested to see what Chemical processes they 
could perform. This revealed that mitochondria are generators of Chemi¬ 
cal energy for the cell. They hamess the energy from the oxidation of food 
molecules, such as sugars, to produce adenosine triphosphate, or ATP— 
the basic Chemical fuel that powers most of the cell's activities. Because 
the mitochondrion consumes oxygen and releases carbón dioxide in the 
course of this activity, the entire process is called cellular respiration — 
essentially, breathing on a cellular level. Without mitochondria, animáis, 
fungi, and plañís would be unable to use oxygen to extract the energy 
they need from the food molecules that nourish them. The process of cel¬ 
lular respiration is considered in detail in Chapter 14. 

outer membrane inner membrane 

Figure 1-17 Mitochondria have a distinctive structure. (A) An electrón micrograph of a cross section of a mitochondrion reveáis 
the extenslve ¡nfolding of the Inner membrane. (B) This three-dimensional representation of the arrangement of the mitochondhal 
membranes shows the smooth outer membrane [gray) and the highly convoluted inner membrane (red). The inner membrane contains 
most of the proteins responsible for cellular respiration—one of the mitochondrion's main functions—and it is highly folded to provide 
a large surface area for this activity. (C) In this schematic cell, the interior space of the mitochondrion is colored orange. (A, courtesy of 
Daniel S. Friend.) 

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CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-18 Mitochondria most likely 
evolved from engulfed bacteria. It ¡s 

virtually certain that mitochondria origínate 
from bacteria that were engulfed by an 
ancestral pre-eukaryotic cell and survived 
inside it, living in symbiosis with their host. 
Note that the double membrane of present- 
day mitochondria is thought to have been 
derived from the plasma membrane and 
outer membrane of the engulfed bacterium. 

Mitochondria contain their own DNA and reproduce by dividing in two. 
Because they resemble bacteria in so many ways, they are thought to 
have been derived from bacteria that were engulfed by some ancestor 
of present-day eukaryotic cells (Figure 1-18). This evidently created a 
symbiotic relationship in which the host eukaryote and the engulfed bac¬ 
terium helped one another to survive and reproduce. 

Chloroplasts Capture Energy from Sunlight 

Chloroplasts are large, green organdíes that are found only in the cells 
of plants and algae, not in the cells of animáis or fungi. These organdíes 
have an even more complex structure than mitochondria: in addition to 
their two surrounding membranes, they possess internal stacks of mem- 
branes containing the green pigment chlorophyll (Figure 1-19). 

Chloroplasts carry out photosynthesis—trapping the energy of sun¬ 
light in their chlorophyll molecules and using this energy to drive the 
manufacture of energy-rich sugar molecules. In the process, they release 

Figure 1-19 Chloroplasts ¡n plant cells 
capture the energy of sunlight. 

(A) A single cell isolated from a leaf 
of a flowering plant, seen in the light 
microscope, showing many green 
chloroplasts. (B) A drawing of one of the 
chloroplasts, showing the inner and outer 
membranes, as well as the highly folded 
System of ¡nternal membranes containing 
the green chlorophyll molecules that absorb 
light energy. (A, courtesy of Preeti Dahiya.) 

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The Eukaryotic Cell 






eukaryotic cell 
capable of 


oxygen as a molecular by-product. Plant cells can then extract this stored 
Chemical energy when they need it, by oxidizing these sugars in their 
mitochondria, just as animal cells do. Chloroplasts thus enable plants to 
get their energy directly from sunlight. And they allow plants to produce 
the food molecules—and the oxygen—that mitochondria use to generate 
Chemical energy in the form of ATP. How these organdíes work together 
is discussed in Chapter 14. 

Like mitochondria, chloroplasts contain their own DNA, reproduce 
by dividing in two, and are thought to have evolved from bacteria—in 
this case, from photosynthetic bacteria that were engulfed by an early 
eukaryotic cell (Figure 1-20). 

Internal Membranes Create Intracellular Compartments 
with Different Functions 

Nuclei, mitochondria, and chloroplasts are not the only membrane- 
enclosed organdíes inside eukaryotic cells. The cytoplasm contains a 
profusión of other organdíes that are surrounded by single membranes 
(see Figure 1-7A). Most of these structures are involved with the cell's 
ability to import raw materials and to export both the useful substances 
and waste products that are produced by the cell. 

The endoplasmic reticulum (ER) is an irregular maze of interconnected 
spaces enclosed by a membrane (Figure 1-21). It is the site where most 
cell-membrane components, as well as materials destined for export 
from the cell, are made. This organelle is enormously enlarged in cells 
that are specialized for the secretion of proteins. Stacks of flattened, 
membrane-enclosed sacs constitute the Golgi apparatus (Figure 1-22), 
which modifies and packages molecules made in the ER that are destined 
to be either secreted from the cell or transported to another cell com- 
partment. Lysosomes are small, irregularly shaped organelles in which 
intracellular digestión occurs, releasing nutrients from ingested food par- 
ticles and breaking down unwanted molecules for either recycling within 
the cell or excretion from the cell. Indeed, many of the large and small 
molecules within the cell are constantly being broken down and remade. 
Peroxisomes are small, membrane-enclosed vesicles that provide a safe 
environment for a variety of reactions in which hydrogen peroxide is 
used to inactivate toxic molecules. Membranes also form many different 
types of small transpon vesicles that ferry materials between one mem¬ 
brane-enclosed organelle and another. All of these membrane-enclosed 
organelles are sketched in Figure 1-23A. 

Figure 1-20 Chloroplasts almost certainly 
evolved from engulfed photosynthetic 
bacteria. The bacteria are thought to have 
been taken up by early eukaryotic cells that 
already contained mitochondria. 

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CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-21 The endoplasmic reticulum 
produces many of the components 
of a eukaryotic cell. (A) Schematic 
diagram of an animal cell shows the 
endoplasmic reticulum (ER) in green. 

(B) Electron micrograph of a thin section 
of a mammalian pancreatic cell shows a 
small part of the ER, of which there are 
vast amounts in this cell type, which ¡s 
specialized for protein secretion. Note that 
the ER is continuous with the membranes 
of the nuclear envelope. The black particles 
studding the particular región of the ER 
shown here are ribosomes, structures that 
transíate RNAs into proteins. Because of its 
appearance, ribosome-coated ER is often 
called "rough ER" to distinguish itfrom 
the "smooth ER," which does not have 
ribosomes bound to it. (B, courtesy of 
Lelio Orci.) 

A continual exchange of materials takes place between the endoplasmic 
reticulum, the Golgi apparatus, the lysosomes, and the outside of the cell. 
The exchange is mediated by transport vesicles that pinch off from the 
membrane of one organelle and fuse with another, like tiny soap bubbles 
budding from and rejoining larger bubbles. At the surface of the cell, for 
example, portions of the plasma membrane tuck inward and pinch off 
to form vesicles that cariy material captured from the external médium 
into the cell—a process called endocytosis (Figure 1-24). Animal cells can 

Figure 1-22 The Golgi apparatus is 
composed of a stack of flattened dises. 

(A) Schematic diagram of an animal cell with 
the Golgi apparatus colored red. (B) More 
realistic drawing of the Golgi apparatus. 
Some of the vesicles seen nearby have 
pinched off from the Golgi stack; others are 
destined to fuse with it. Only one stack is 
shown here, but several can be present in a 
cell. (C) Electron micrograph that shows the 
Golgi apparatus from a typical animal cell. 
(C, courtesy of Brij J. Gupta.) 

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The Eukaryotic Cell 


Figure 1-23 Membrane-enclosed 
organelles are distributed throughout 
the eukaryotic cell cytoplasm. (A) The 

membrane-enclosed organelles, shown 
in different colors, are each speclallzed 
to perform a different function. (B) The 
cytoplasm that filis the space outslde 
of these organelles ¡s called the cytosol 
(colored b/ue). 

engulf very large partióles, or even entire foreign cells, by endocytosis. In 
the reverse process, called exocytosis, vesicles from inside the cell fuse 
with the plasma membrane and release their contents into the external 
médium (see Figure 1-24); most of the hormones and signal molecules 
that allow cells to communicate with one another are secreted from cells 
by exocytosis. How membrane-enclosed organelles move proteins and 
other molecules from place to place inside the cell is discussed in detail 
in Chapter 15. 

The Cytosol Is a Concentrated Aqueous Gel of Large and 
Small Molecules 

If we were to strip the plasma membrane from a eukaryotic cell and then 
remove all of its membrane-enclosed organelles, including the nucleus, 
endoplasmic reticulum, Golgi apparatus, mitochondria, chloroplasts, and 
so on, we would be left with the cytosol (see Figure 1-23B). In other 
words, the cytosol is the part of the cytoplasm that is not contained within 
intracellular membranes. In most cells, the cytosol is the largest single 
compartment. It contains a host of large and small molecules, crowded 
together so closely that it behaves more like a water-based gel than a 
liquid solution (Figure 1-25). The cytosol is the site of many Chemical 
reactions that are fundamental to the cell’s existence. The early steps in 
the breakdown of nutrient molecules take place in the cytosol, for exam- 
ple, and it is here that most proteins are made by ribosomes. 

The Cytoskeleton Is Responsible for Directed Cell 

The cytoplasm is notjust a structureless soup of Chemicals and organelles. 
Using an electrón microscope, one can see that in eukaryotic cells the 
cytosol is criss-crossed by long, fine filaments. Frequently, the filaments 
are seen to be anchored at one end to the plasma membrane or to radí¬ 
ate out from a central site adjacent to the nucleus. This System of protein 
filaments, called the cytoskeleton, is composed of three major filament 
types (Figure 1-26). The thinnest of these filaments are the actin fila¬ 
mente-, they are abundant in all eukaryotic cells but occur in especially 
large numbers inside muscle cells, where they serve as a central part 
of the machinery responsible for muscle contraction. The thickest fila¬ 
ments in the cytosol are called microtubules, because they have the form 
of minute hollow tubes. In dividing cells, they become reorganized into a 
spectacular array that helps pulí the duplicated chromosomes in opposite 



Figure 1-24 Eukaryotic cells engage in 
continual endocytosis and exocytosis. 

They import extracellular materials by 
endocytosis and secrete Intracellular 
materials by exocytosis. 

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22 CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-25 The cytoplasm is stuffed with 
organelles and a host of large and small 
molecules. This schematic drawing, which 
extends across two pages and is based 
on the known sizes and concentrations 
of molecules in the cytosol, shows how 
crowded the cytoplasm is. Proteins are blue, 
membrane lipids are yellow, and ribosomes 
and DNA are pink. The panorama begins on 
the far left atthe plasma membrane, moves 
through the endoplasmic reticulum, Golgi 
apparatus, and a mitochondrion, and ends 
on the far right in the nucleus. (Courtesy of 
D. Goodsell.) 


Suggest a reason why it would be 
advantageous for eukaryotic cells to 
evolve elabórate internal membrane 
systems that allow them to import 
substances from the outside, as 
shown in Figure 1-24. 

directions and distribute them equally to the two daughter cells (Figure 
1-27). Intermedíate in thickness between actin fllaments and microtu- 
bules are the intermedíate Jilaments, which serve to strengthen the cell. 
These three types of filaments, together with other proteins that attach to 
them, form a system of girders, ropes, and motors that gives the cell its 
mechanical strength, Controls its shape, and drives and guides its move- 
ments (Movie 1.2 and Movie 1.3). 

Because the cytoskeleton governs the intemal organization of the cell 
as well as its external features, it is as necessaiy to a plant cell—boxed 
in by a tough wall of extracellular matrix—as it is to an animal cell that 
freely bends, stretches, swims, or crawls. In a plant cell, for example, 
organelles such as mitochondria are driven in a constant stream around 
the cell interior along cytoskeletal tracks (Movie 1.4). And animal cells 
and plant cells alike depend on the cytoskeleton to sepárate their intemal 
components into two daughter cells during cell división (see Figure 1-27). 
The cytoskeleton's role in cell división may be its most ancient func- 
tion. Even bacteria contain proteins that are distantly related to those of 
eukaryotic actin fllaments and microtubules, forming filaments that play 
a part in prokaryotic cell división. We examine the cytoskeleton in detail 
in Chapter 17, discuss its role in cell división in Chapter 18, and review 
how it responds to signáis from outside the cell in Chapter 16. 

The Cytoplasm Is Far from Static 

The cell interior is in constant motion. The cytoskeleton is a dynamic 
jungle of protein ropes that are continually being strung together and 
taken apart; its fllaments can assemble and then disappear in a matter 
of minutes. Motor proteins use the energy stored in molecules of ATP to 
trundle along these tracks and cables, carrying organelles and proteins 
throughout the cytoplasm, and racing across the width of the cell in sec- 
onds. In addition, the large and small molecules that fill every free space 
in the cell are swept to and fro by random thermal motion, constantly 
colliding with one another and with other structures in the cell’s crowded 
cytoplasm (Movie 1-5). 

Figure 1-26 The cytoskeleton is a 
network of protein filaments that criss- 
crosses the cytoplasm of eukaryotic 
cells. The three major types of filaments 
can be detected using different fluorescent 
stains. Shown here are (A) actin filaments, 
(B) microtubules, and (C) intermedíate 
filaments. (A, courtesy of Simón Barry 
and Chris D'Lacey; B, courtesy of Nancy 
Kedersha; C, courtesy of Clive Lloyd.) 

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The Eukaryotic Cell 


Of course, neither the bustling nature of the cell's interior ñor the details 
of cell structure were appreciated when scientists first peered at cells in 
a microscope; our knowledge of cell structure accumulated slowly. A few 
of the key discoveries are listed in Table 1-1 . In addition, Panel 1-2 sum- 
marizes the differences between animal, plant, and bacterial cells. 

Eukaryotic Cells May Have Originated as Predators 

Eukaryotic cells are typically 10 times the length and 1000 times the vol- 
ume of prokaryotic cells, although there is huge size variation within 
each category. They also possess a whole collection of features—a 
cytoskeleton, mitochondria, and other organdíes—that set them apart 
from bacteria and archaea. 

When and how eukaiyotes evolved these systems remains something of a 
mystery. Although eukaryotes, bacteria, and archaea must have diverged 
from one another veiy early in the histoiy of life on Earth (discussed in 
Chapter 14), the eukaiyotes did not acquire all of their distinctive features 
at the same time (Figure 1-28). According to one theory, the ancestral 
eukaryotic cell was a predator that fed by capturing other cells. Such a 
way of life requires a large size, a flexible membrane, and a cytoskel¬ 
eton to help the cell move and eat. The nuclear compartment may have 
evolved to keep the DNA segregated from this physical and Chemical 
hurly-burly, so as to allow more delicate and complex control of the way 
the cell reads out its genetic information. 

Such a primitive cell, witha nucleus and cytoskeleton, was most likely 
the sort of cell that engulfed the free-living, oxygen-consuming bacte¬ 
ria that were the likely ancestors of the mitochondria (see Figure 1-18). 
This partnership is thought to have been established 1.5 billion years ago, 
when the Earth's atmosphere first became rich in oxygen. A subset of 


Discuss the relative advantages 
and disadvantages of light and 
electrón microscopy. How could 
you best visualize (a) a living skin 
cell, (b) a yeast mitochondrion, (c) a 
bacterium, and (d) a microtubule? 

Figure 1-27 Microtubules help distribute 
the chromosomes ¡n a dividing cell. 

When a cell divides, its nuclear envelope 
breaks down and its DNA condenses into 
visible chromosomes, each of which has 
duplicated to form a pair of conjoined 
chromosomes that will ultimately be pulled 
apart into sepárate cells by microtubules. In 
the transmission electrón micrograph (left), 
the microtubules are seen to radíate from 
foci at opposite ends of the dividing cell. 
(Photomicrograph courtesy of 
Conly L. Rieder.) 

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24 CHAPTER 1 Cells: The Fundamental Units of Life 

1 TABLE 1-1 t 



Hooke uses a primitive microscope to describe small chambers in sections of corkthat he calis "cells." 


Leeuwenhoek reports his discovery of protozoa. Nine years later, he sees bacteria for the first time. 


Brown publishes his microscopic observations of orchids, clearly describing the cell nucleus. 


Schleiden and Schwann propose the cell theory, stating that the nucleated cell is the universal building block of plant 
and animal tissues. 


Kólliker describes mitochondria in muscle cells. 


Flemming describes with great clarity chromosome behavior during mitosis in animal cells. 


Cajal and other histologists develop staining methods that reveal the structure of nerve cells and the organization of 
neural tissue. 


Golgi first sees and describes the Golgi apparatus by staining cells with silver nitrate. 


Boveri links chromosomes and heredity by observing chromosome behavior during sexual reproduction. 


Palade, Porter, and Sjóstrand develop methods of electrón microscopy that enable many intracellular structures to be 
seen for the first time. In one of the first applications of these techniques, Huxley shows that muscle contains arrays of 
protein filaments—the first evidence of a cytoskeleton. 


Robertson describes the bilayer structure of the cell membrane, seen for the first time in the electrón microscope. 


Kendrew describes the first detailed protein structure (sperm whale myoglobin) to a resolution of 0.2 nm using X-ray 
crystallography. Perutz proposes a lower-resolution structure for hemoglobin. 


Christian de Duve and his colleagues use a cell-fractionation technique to sepárate peroxisomes, mitochondria, and 
lysosomes from a preparation of rat liver. 


Petran and collaborators make the first confocal microscope. 


Frye and Edidin use fluorescent antibodies to show that plasma membrane molecules can diffuse in the plañe of the 
membrane, indicating that cell membranes are fluid. 


Lazarides and Weber use fluorescent antibodies to stain the cytoskeleton. 


Chalfie and collaborators introduce green fluorescent protein (GFP) as a marker to follow the behavior of proteins in 
living cells. 

these cells later acquired chloroplasts by engulfing photosynthetic bacte¬ 
ria (see Figure 1-20). The likely history of these endosymbiotic events is 
illustrated in Figure 1-28. 

That single-celled eukaryotes can prey upon and swallow other cells 
is borne out by the behavior of many of the free-living, actively motile 

Figure 1-28 Where did eukaryotes 
come from? The eukaryotic, bacterial, 
and archaean lineages diverged from one 
another very early in the evolution of life 
on Earth. Some time later, eukaryotes are 
thought to have acquired mitochondria; 
later still, a subset of eukaryotes acquired 
chloroplasts. Mitochondria are essentially 
the same in plants, animáis, and fungí, and 
therefore were presumably acquired before 
these lines diverged. 

LibertadDigital \ 2015 




extracellular matrix 

chromatin (DNA) 

nuclear pore 

nuclear envelope 


Three cell types are drawn 
here in a more realistic 
manner than ¡n the schematic 
drawing in Figure 1-23. The 
same colors are used, however, 
to distinguish the organelles 
of the cell. The animal cell 
drawing is based on a 
fibroblast, a cell that 
inhabits connective tissue 
and deposits extracellular 
matrix. A micrograph of a 
living fibroblast is shown in 
Figure 1-6A. The plant cell 
drawing is typical of a young 
leaf cell. The bacterium shown 
is rod-shaped and has a single 
flagellum for motility; note its 
much smaller size (compare 
scale bars). 

• j. plasma membrane 


vacuole membrane (tonoplast) 


CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-29 One protozoan eats another. 

(A) The scanning electrón micrograph shows 
Didinium on its own, with ¡ts circumferential 
rings of beating cilia and its "snout" at the 
top. (B) Didinium ¡s seen ingesting another 
ciliated protozoan, a Paramecium. (Courtesy 
of D. Barlow.) 

microorganisms called protozoans. Didinium, for example, is a large, 
carnivorous protozoan with a diameter of about 150 pm—roughly 10 
times that of the average human cell. It has a globular body encircled by 
two fringes of cilia, and its front end is flattened except for a single pro- 
trusion rather like a snout (Figure 1-29A). Didinium swims at high speed 
by means of its beating cilia. When it encounters a suitable prey, usually 
another type of protozoan, it releases numerous small, paralyzing darts 
from its snout región. Didinium then attaches to and devours the other 
cell, inverting like a hollow ball to engulf its victim, which can be almost 
as large as itself (Figure 1-29B). 

Not all protozoans are predators. They can be photosynthetic or carnivo¬ 
rous, motile or sedentary. Their anatomy is often elabórate and ineludes 
such structures as sensoiy bristles, photoreceptors, beating cilia, stalk- 
like appendages, mouthparts, stinging darts, and musclelike contractile 
bundles (Figure 1-30). Although they are single cells, protozoans can be 
as intricate and versatile as many multicellular organisms. Much remains 
to be learned about fundamental cell biology from studies of these fasci- 
nating life-forms. 


All cells are thought to be descended from a common ancestor, whose 
fundamental properties have been conserved through evolution. Thus 
knowledge gained from the study of one organism contributes to our 
understanding of others, including ourselves. But certain organisms are 
easier than others to study in the laboratoiy. Some reproduce rapidly and 
are convenient for genetic manipulations; others are multicellular but 
transparent, so that one can directly watch the development of all their 
internal tissues and organs. For reasons such as these, large communi- 
ties of biologists have become dedicated to studying different aspeets of 
the biology of a few chosen species, pooling their knowledge to gain a 
deeper understanding than could be achieved if their efforts were spread 
over many different species. Although the roster of these representa- 
tive organisms is continually expanding, a few stand out in terms of the 
breadth and depth of information that has been accumulated about them 
over the years—knowledge that contributes to our understanding of how 
all cells work. In this section, we examine some of these model organ¬ 
isms and review the benefits that each offers to the study of cell biology 
and, in many cases, to the promotion of human health. 

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Model Organis 


Figure 1-30 An assortment of protozoans illustrates the enormous variety within this class of single-celied 
microorganisms. These drawings are done to different scales, but ¡n each case the scale bar represents 
10 |tm. The organisms in (A), (C), and (G) are ciliates; (B) ¡s a heliozoan; (D) is an amoeba; (E) ¡s a dinoflagellate; and 
(F) ¡s a euglenoid. To see the latter ¡n action, watch Movie 1.6. (From M.A. Sleigh, The Biology of Protozoa. London: 
Edward Arnold, 1973. With permission from Edward Arnold.) 

Molecular Biologists Have Focused on E. coli 

In molecular terms, we understand the workings of the bacterium 
Escherichia coli—E. coli for short—more thoroughly than those of any 
other living organism (see Figure 1-10). This small, rod-shaped cell nor- 
mally lives in the gut of humans and other vertebrates, but it also grows 
happily and reproduces rapidly in a simple nutrient broth in a culture 

Most of our knowledge of the fundamental mechanisms of life—including 
how cells replícate their DNA and how they decode these genetic instruc- 
tions to make proteins—has come from studies of E. coli. Subsequent 
research has conflrmed that these basic processes occur in essentially the 
same way in our own cells as they do in E. coli. 

Brewer's Yeast Is a Simple Eukaryotic Cell 

We tend to be preoccupied with eukaryotes because we are eukary- 
otes ourselves. But human cells are complicated and reproduce relatively 
slowly. To get a handle on the fundamental biology of eukaryotic cells, 
it is often advantageous to study a simpler cell that reproduces more 
rapidly. A popular choice has been the budding yeast Saccharomyces 
cerevisiae (Figure 1-31) —the same microorganism that is used for brew- 
ing beer and baking bread. 

5. cerevisiae is a small, single-celied fungus that is at least as closely 
related to animáis as it is to plants. Like other fungí, it has a rigid cell wall, 
is relatively immobile, and possesses mitochondria but not chloroplasts. 
When nutrients are plentiful, S. cerevisiae reproduces almost as rapidly as 
a bacterium. Yet it carries out all the basic tasks that every eukaryotic cell 
must perform. Genetic and biochemical studies in yeast have been crucial 
to understanding many basic mechanisms in eukaryotic cells, including 
the cell-division cycle—the chain of events by which the nucleus and all 
the other components of a cell are duplicated and parceled out to create 
two daughter cells. The machinery that governs cell división has been 


Your next-door neighbor has 
donated $100 in support of cáncer 
research and is horrified to learn 
that her money is being spent on 
studying brewer's yeast. How could 
you put her mind at ease? 

LibertadDigital \ 2015 


CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-31 The yeast Saccharomyces cerevisiae ¡s a model 
eukaryote. In this scanning electrón micrograph, a few yeast cells are 
seen in the process of dividing, which they do by budding. Another 
micrograph of the same species is shown in Figure 1-13. (Courtesy of 
Ira Herskowitz and Eric Schabatach.) 

so well conserved over the course of evolution that many of its compo- 
nents can function interchangeably in yeast and human cells (see How 
We Know, pp. 30-31). Darwin himselfwould no doubt have been stunned 
by this dramatic example of evolutionary conservation. 

Arabidopsis Has Been Chosen as a Model Plant 

The large multicellular organisms that we see around us—both plants 
and animáis—seem fantastically varied, but they are much closer to one 
another in their evolutionary origins, and more similar in their basic 
cell biology, than the great host of microscopic single-celled organisms. 
Whereas bacteria, archaea, and eukaryotes separated frorn each other 
more than 3 billion years ago, plants, animáis, and fungí diverged only 
about 1.5 billion years ago, and the different species of flowering plants 
less than 200 million years ago. 

The cióse evolutionary relationship among all flowering plants means 
that we can gain insight into their cell and molecular biology by focusing 
on just a few convenient species for detailed analysis. Out of the several 
hundred thousand species of flowering plants on Earth today, molecular 
biologists have focused their efforts on a small weed, the common wall 
cress Arabidopsis thaliana (Figure 1-32), which can be grown indoors 
in large numbers: one plant can produce thousands of offspring within 
8-10 weeks. Because genes found in Arabidopsis have counterparts in 
agricultural species, studying this simple weed provides insights into 
the development and physiology of the crop plants upon which our lives 
depend, as well as into the evolution of all the other plant species that 
domínate nearly every ecosystem on Earth. 

Model Animáis Inelude Flies, Fish, Worms, and Mice 

Multicellular animáis account for the majority of all named species of 
living organisms, and the majority of animal species are inseets. It is fit- 
ting, therefore, that an insect, the small fruit fly Drosophila melanogaster 
(Figure 1-33), should occupy a central place in biological research. In 
fact, the foundations of classical genetics were built to a large extent on 
studies of this insect. More than 80 years ago, genetic analysis of the fruit 
fly provided definitive proof that genes—the units of heredity—are car- 
ried on chromosomes. In more recent times, Drosophila, more than any 
other organism, has shown us how the genetic instructions encoded in 
DNA molecules direct the development of a fertilized egg cell (or zygote) 
into an adult multicellular organism containing vast numbers of different 
cell types organized in a precise and predictable way. Drosophila mutants 
with body parís strangely misplaced or oddly pattemed have provided 
the key to identifying and characterizing the genes that are needed to 
make a properly structured adult body, with gut, wings, legs, eyes, and 
all the other bits and pieces in their correct places. These genes—which 
are copied and passed on to every cell in the body—define how each cell 
will behave in its social interactions with its sisters and cousins, thus 
controlling the structures that the cells can create. Moreover, the genes 

Figure 1-32 Arabidopsis thaliana, the common wall cress, is a 
model plant. This small weed has become the favorite organism 
of plant molecular and developmental biologists. (Courtesy of Toni 
Hayden and the John Innes Centre.) 

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Model Organisms 


Figure 1-33 Drosophila melanogaster is a 
favorite among developmental biologists 
and geneticists. Molecular genetic studies 
on this small fly have provlded a key to the 
understanding of how all animáis develop. 
(Courtesy of E.B. Lewis.) 

responsible for the development of Drosophila have turned out to be 
amazingly similar to those of humans—far more similar than one would 
suspect from outward appearances. Thus the fly serves as a valuable 
model for studying human development and disease. 

Another widely studied organism is the nematode worm Caenorhabditis 
elegans (Figure 1-34), a harmless relative of the eelworms that attack the 
roots of crops. Smaller and simpler than Drosophila, this creature devel- 
ops with clockwork precisión from a fertilized egg cell into an adult that 
has exactly 959 body cells (plus a variable number of egg and sperm 
cells)—an unusual degree of regularity for an animal. We now have a 
minutely detailed description of the sequence of events by which this 
occurs—as the cells divide, move, and become specialized according to 
strict and predictable rules. And a wealth of mutants are available for 
testing how the worm’s genes direct this developmental ballet. Some 70% 
of human genes have some counterpart in the worm, and C. elegans, like 
Drosophila, has proved to be a valuable model for many of the devel¬ 
opmental processes that occur in our own bodies. Studies of nematode 
development, for example, have led to a detailed molecular understand¬ 
ing of apoptosis, a form of programmed cell death by which surplus cells 
are disposed of in all animáis—a topic of great importance for cáncer 
research (discussed in Chapters 18 and 20). 

Another organism that is providing molecular insights into developmen¬ 
tal processes, particularly in vertebrates, is the zebrajish. Because this 

Figure 1-34 Caenorhabditis elegans is a small nematode worm that normally 
lives in the soil. Most individuáis are hermaphrodltes, producing both sperm and 
eggs (the latter of which can be seen along the underside of the animal). C. elegans 
was the first multicellular organism to have its complete genome sequenced. 
(Courtesy of María Gallegos.) 

LibertadDigital \ 2015 




All living things are made of cells, and all cells—as we 
have discussed in this chapter—are fundamentally simi¬ 
lar inside: they store their genetic instructions in DNA 
molecules, which direct the production of RNA mol- 
ecules, which in turn direct the production of proteins. 
It is largely the proteins that carry out the cell's Chemi¬ 
cal reactions, give the cell its shape, and control its 
behavior. But how deep do these similarities between 
cells—and the organisms they comprise—really run? 
Are parts from one organism interchangeable with parts 
frorn another? Would an enzyme that breaks down glu- 
cose in a bacterium be able to digest the same sugar if it 
were placed inside a yeast cell or a cell from a lobster or 
a human? What about the molecular machines that copy 
and interpret genetic information? Are they functionally 
equivalent from one organism to another? Insights have 
come from many sources, but the most stunning and 
dramatic answer carne from experiments performed on 
humble yeast cells. These studies, which shocked the 
biological community, focused on one of the most fun¬ 
damental processes of life—cell división. 

División and discovery 

All cells come from other cells, and the only way to 
make a new cell is through división of a preexisting 
one. To reproduce, a parent cell must execute an orderly 
sequence of reactions, through which it duplicates its 
contents and divides in two. This critical process of 
duplication and división—known as the cell-division 
cycle, or cell cycle for short—is complex and carefully 
controlled. Defects in any of the proteins involved can 
be devastating to the cell. 

Fortunately for biologists, this acute rebanee on cru¬ 
cial proteins makes them easy to identify and study. If a 
protein is essential for a given process, a mutation that 
results in an abnormal protein—or in no protein at all— 
can prevent the cell from carrying out the process. By 
isolating organisms that are defective in their cell-divi- 
sion cycle, scientists have worked backward to discover 
the proteins that control progress through the cycle. 

The study of cell-cycle mutants has been particularly 
successful in yeasts. Yeasts are unicellular fungí and are 
popular organisms for such genetic studies. They are 
eukaryotes, like us, but they are small, simple, rapidly 
reproducing, and easy to manipúlate genetically. Yeast 
mutants that are defective in their ability to complete 
cell división have led to the discoveiy of many genes 
that control the cell-division cycle—the so-called Cdc 
genes—and have provided a detailed understanding of 
how these genes, and the proteins they encode, actually 

Paul Nurse and his colleagues used this approach to 
identify Cdc genes in the yeast Schizosaccharomyces 
pombe, which is named after the African beer from 
which it was first isolated. S. pombe is a rod-shaped cell, 
which grows by elongation at its ends and divides by fis- 
sion into two, through the formation of a partition in the 
center of the rod. The researchers found that one of the 
Cdc genes they had identifled, called Cdc2, was required 
to trigger several key events in the cell-division cycle. 
When that gene was inactivated by a mutation, the yeast 
cells would not divide. And when the cells were pro¬ 
vided with a normal copy of the gene, their ability to 
reproduce was restored. 

It's obvious that replacing a faulty Cdc2 gene in S. pombe 
with a functioning Cdc2 gene from the same yeast 
should repair the damage and enable the cell to divide 
normally. But what about using a similar cell-division 
gene from a different organism? That's the question the 
Nurse team tackled next. 

Next of kin 

Saccharomyces cerevisiae is another kind of yeast and 
is one of a handful of model organisms biologists have 
chosen to study to expand their understanding of how 
cells work. Also used to brew beer, S. cerevisiae divides 
by forming a small bud that grows steadily until it sepa- 
rates from the mother cell (see Figures 1-13 and 1-31). 
Although S. cerevisiae and S. pombe differ in their style of 
división, both rely on a complex network of interacting 
proteins to get the job done. But could the proteins from 
one type of yeast substitute for those of the other? 

To find out, Nurse and his colleagues prepared DNA 
from healthy S. cerevisiae, and they introduced this DNA 
into S. pombe cells that contained a mutation in the Cdc2 
gene that kept the cells from dividing when the tem¬ 
peratura was elevated. And they found that some of the 
mutant S. pombe cells regained the ability to probferate 
when warm. If spread onto a culture píate containing 
a growth médium, the rescued cells could divide again 
and again to form visible colonies, each containing mil- 
lions of individual yeast cells (Figure 1-35). Upon closer 
examination, the researchers discovered that these "res¬ 
cued'' yeast cells had received a fragment of DNA that 
contained the S. cerevisiae versión of Cdc2— a gene that 
had been discovered in pioneering studies of the cell 
cycle by Lee Hartwell and colleagues. 

The result was exciting, but perhaps not all that sur- 
prising. After all, how different can one yeast be from 
another? A more demanding test would be to use DNA 
from a more distant relative. So Nurse's team repeated 
the experiment, this time using human DNA. And the 
results were the same. The human equivalent of the 

ÁbertadDigital \ 2015 

Model Organisms 


substitute for the Cdc2 gene will 
divide to form a colony 
at the warm temperature 

Figure 1-35 S. pombe mutants defective in a cell-cycle gene 
can be rescued by the equivalent gene from S. cerevisiae. 

DNA ¡s collected from S. cerevisiae and broken into large 
fragments, which are introduced into a culture of mutant 
S. pombe cells dividing at room temperature. We discuss how 
DNA can be manipulated and transferred into different cell types 
in Chapter 10. These yeast cells are then spread onto a píate 
containing a suitable growth médium and are ¡ncubated at a 
warm temperature, at which the mutant Cdc2 protein is inactive. 
The rare cells that survive and proliferate on these plates have 
been rescued by ¡ncorporation of a foreign gene that allows 
them to divide normally at the higher temperature. 

S. pombe Cdc2 gene could rescue the mutant yeast cells, 
allowing them to divide normally. 

Gene reading 

This result was much more surprising—even to Nurse. 
The ancestors of yeast and humans diverged some 1.5 
billion years ago. So it was hard to believe that these 

two organisms would orchestrate cell división in such 
a similar way. But the results clearly showed that the 
human and yeast proteins are functionally equivalent. 
Indeed, Nurse and colleagues demonstrated that the 
proteins are almost exactly the same size and consist of 
amino acids strung together in a very similar order; the 
human Cdc2 protein is identical to the S. pombe Cdc2 
protein in 63% of its amino acids and is identical to the 
equivalent protein from S. cerevisiae in 58% of its amino 
acids (Figure 1-36). Together with Tim Hunt, who dis- 
covered a different cell-cycle protein called cyclin, Nurse 
and Hartwell shared a 2001 Nobel Prize for their studies 
of key regulators of the cell cycle. 

The Nurse experiments showed that proteins from very 
different eukaiyotes can be functionally interchange- 
able and suggested that the cell cycle is controlled in 
a similar fashion in every eukaiyotic organism alive 
today. Apparently, the proteins that orchestrate the cycle 
in eukaiyotes are so fundamentally important that they 
have been conserved almost unchanged over more than 
a billion years of eukaiyotic evolution. 

The same experiment also highlights another, even more 
basic, point. The mutant yeast cells were rescued, not by 
direct injection of the human protein, but by introduc- 
tion of a piece of human DNA. Thus the yeast cells could 
read and use this information correctly, indicating that, 
in eukaiyotes, the molecular machinery for reading the 
information encoded in DNA is also similar from cell to 
cell and from organism to organism. A yeast cell has 
all the equipment it needs to interpret the instructions 
encoded in a human gene and to use that information to 
direct the production of a fully functional human protein. 
The story of Cdc2 is just one of thousands of examples of 
how research in yeast cells has provided critical insights 
into human biology. Although it may sound paradoxi- 
cal, the shortest, most efficient path to improving human 
health will often begin with detailed studies of the biol¬ 
ogy of simple organisms such as brewer's or baker's 



S. cerevisiae •••fglarafgvplraytheivtlwyrapevllggkqystgvdtwsigcifaehcnrlpifsgdseidqifkiprvlgtpneaiwpdivylpdfkpsfp... 

Figure 1-36 The cell-division-cycle proteins from yeasts and human are very similar in their amino acid sequences. Identities 
between the amino acid sequences of a región of the human Cdc2 protein and a similar región of the equivalent proteins in 
S. pombe and S. cerevisiae are ¡ndicated by green shading. Each amino acid is represented by a single letter. 


CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-37 Zebrafish are popular models for studies of vertébrate 
development. (A) These small, hardy, tropical fish are a staple ¡n many 
home aquaria. But they are also ideal for developmental studies, as their 
transparent embryos (B) make it easy to observe cells moving and changing 
their characters ¡n the living organism as it develops. (A, courtesy of Steve 
Baskauf; B, from M. Rhinn et al., Neural Dev. 4:12, 2009. With permission 
from BioMed Central Ltd.) 

creature is transparent for the first 2 weeks of its life, it provides an ideal 
System in which to observe how cells behave during development in a 
living animal (Figure 1-37). 

Mammals are among the most complex of animáis, and the mouse has 
long been used as the model organism in which to study mammalian 
genetics, development, immunology, and cell biology. Thanks to modern 
molecular biological techniques, it is now possible to breed mice with 
deliberately engineered mutations in any specific gene, or with artificially 
constructed genes introduced into them. In this way, one can test what 
a given gene is required for and how it functions. Almost every human 
gene has a counterpart in the mouse, with a similar DNA sequence and 
function. Thus, this animal has proven an excellent model for studying 
genes that are important in both human health and disease. 

Biologists Also Directly Study Human Beings and 
Their Cells 

Humans are not mice—or fish or flies or worms or yeast—and so we also 
study human beings themselves. Like bacteria or yeast, our individual 
cells can be harvested and grown in culture, where we can study their 
biology and more closely examine the genes that govern their functions. 
Given the appropriate surroundings, most human cells—indeed, most 
cells from animáis or plants—will survive, proliferate, and even express 
specialized properties in a culture dish. Experiments using such cultured 
cells are sometimes said to be carried out in vitro (literally, "in glass") to 
contrast them with experiments on intact organisms, which are said to be 
carried out in vivo (literally, "in the living"). 

Although not true for all types of cells, many types of cells grown in 
culture display the differentiated properties appropriate to their origin: 
fibroblasts, a major cell type in connective tissue, continué to secrete 
collagen; cells derived from embryonic skeletal muscle fuse to form 
muscle fibers, which contract spontaneously in the culture dish; nerve 
cells extend axons that are electrically excitable and make synapses with 
other nerve cells; and epithelial cells form extensive sheets, with many 
of the properties of an intact epithelium (Figure 1-38). Because cultured 
cells are maintained in a controlled environment, they are accessible to 
study in ways that are often not possible in vivo. For example, cultured 
cells can be exposed to hormones or growth factors, and the effects that 
these signal molecules have on the shape or behavior of the cells can be 
easily explored. 

In addition to studying human cells in culture, humans are also exam- 
ined directly in clinics. Much of the research on human biology has been 
driven by medical interests, and the medical database on the human spe- 
cies is enormous. Although naturally occurring mutations in any given 
human gene are rare, the consequences of many mutations are well doc- 
umented. This is because humans are unique among animáis in that they 
report and record their own genetic defects: in no other species are bil- 
lions of individuáis so intensively examined, described, and investigated. 
Nevertheless, the extent of our ignorance is still daunting. The mam¬ 
malian body is enormously complex, being formed from thousands of 

LibertadDigital \ 2015 

Model Organis 


billions of cells, and one might despair of ever understanding how the 
DNA in a fertilized mouse egg cell makes it generate a mouse rather than 
a fish, or how the DNA in a human egg cell directs the development of 
a human rather than a mouse. Yet the revelations of molecular biology 
have made the task seem eminently approachable. As much as anything, 
this new optimism has come from the realization that the genes of one 
type of animal have cióse counterparts in most other types of animáis, 
apparently serving similar functions (Figure 1-39). We all have a com- 
mon evolutionary origin, and under the surface it seems that we share 
the same molecular mechanisms. Flies, worms, fish, mice, and humans 
thus provide a key to understanding how animáis in general are made 
and how their cells work. 

Comparing Genome Sequences Reveáis Life's Common 

At a molecular level, evolutionary change has been remarkably slow. We 
can see in present-day organisms many features that have been preserved 
through more than 3 billion years of life on Earth—about one-fifth of the 
age of the universe. This evolutionary conservatism provides the founda- 
tion on which the study of molecular biology is built. To set the scene for 
the chapters that follow, therefore, we end this chapter by considering a 
little more closely the family relationships and basic similarities among 
all living things. This topic has been dramatically clarified in the past few 
years by technological advances that have allowed us to determine the 
complete genome sequences of thousands of organisms, including our 
own species (as discussed in more detail in Chapter 9). 

The first thing we note when we look at an organism's genome is its 
overall size and how many genes it packs into that length of DNA. 
Prokaryotes carry very little superfluous genetic baggage and, nucleotide- 

Figure 1-38 Cells in culture often display 
properties that reflect their origin. 

(A) Phase-contrast micrograph of fibroblasts 
in culture. (B) Micrograph of cultured 
myoblasts, some of which have fused 
to form multinucleate muscle cells that 
spontaneously contract in culture. 

(C) Cultured epithelial cells forming a cell 
sheet. Movie 1.7 shows a single heart 
muscle cell beating in culture. (A, courtesy 
of Daniel Zicha; B, courtesy of Rosalind 
Zalin; C, from K.B. Chua et al., Proc. Nati 
Acad. Sci, USA 104:11424-11429, 2007, with 
permission from the National Academy of 

Figure 1-39 Different species share 
similar genes. The human baby and the 
mouse shown here have similar white 
patches on their foreheads because they 
both have defects in the same gene (called 
Kit), which is required for the development 
and maintenance of some pigment cells. 
(Courtesy of R.A. Fleischman, from Proc. 
Nati Acad. Sci. USA 88:10885-10889, 

1991. With permission from the National 
Academy of Sciences.) 

LibertadDigital \ 2015 


CHAPTER 1 Cells: The Fundamental Units of Life 

Figure 1-40 Organisms vary enormously 
in the size of their genomes. Genome size 
¡s measured ¡n nucleotide pairs of DNA per 
haploid genome, that is, per single copy 
of the genome. (The body cells of sexually 
reproducing organisms such as ourselves 
are generalíy diploid: they contain two 
copies of the genome, one inherited from 
the mother, the other from the father.) 
Closely related organisms can vary widely 
in the quantity of DNA in their genomes (as 
¡ndicated by the length of the green bars), 
even though they contain similar numbers 
of functionally distinct genes. (Adapted from 
T.R. Gregory, 2008, Animal Genome Size 

1 0 5 1 0 6 10 7 10 8 10 9 10 10 10 11 1Q 12 

nucleotide pairs per haploid genome 

for-nucleotide, they squeeze a lot of information into their relatively small 
genomes. E. coli, for example, carries its genetic instructions in a sin¬ 
gle, circular, double-stranded molecule of DNA that contains 4.6 million 
nucleotide pairs and 4300 genes. The simplest known bacterium contains 
only about 500 genes, but most prokaryotes have genomes that contain 
at least 1 million nucleotide pairs and 1000-8000 genes. With these few 
thousand genes, prokaryotes are able to thrive in even the most hostile 
environments on Earth. 

The compact genomes of typical bacteria are dwarfed by the genomes of 
typical eukaryotes. The human genome, for example, contains about 700 
times more DNA than the E. coli genome, and the genome of an amoeba 
contains about 100 times more than ours (Figure 1-40). The rest of the 
model organisms we have described have genomes that fall somewhere 
in between E. coli and human in terms of size. 5. cerevisiae contains about 
2.5 times as much DNA as E. coli-, Drosophila has about 10 times more 
DNA per cell than yeast; and mice have about 20 times more DNA per cell 
than the fruit fly (Table 1-2). 







Genome size* 
(nucleotide pairs) 

number of genes 

Homo sapiens 

3200 x 10 6 


Mus musculus 

2800 x 10 6 


Drosophila melanogaster 
(fruit fly) 

200 x 10 6 


Arabidopsis thaliana 

220 x 10 6 


Caenorhabditis elegans 

130 x 10 6 


Saccharomyces cerevisiae 

13 x 10 6 


Escherichia coli 

4.6 x 10 6 


*Genome size ineludes an estímate for the amount of highly repeated DNA sequence 
not in genome databases. 

LibertadDigital \ 2015 

Chapter 1 Essential Concepts 35 

In terms of gene numbers, however, the differences are not so great. We 
have only about six times as many genes as E. coli. Moreover, many of 
our genes—and the proteins they encode—fall into closely related fam- 
ily groups, such as the family of hemoglobins, which has nine closely 
related members in humans. Thus the number of fundamentally different 
proteins in a human is not veiy many times more than in a bacterium, 
and the number of human genes that have identiflable counterparts in the 
bacterium is a significant fraction of the total. 

This high degree of "family resemblance" is striking when we compare 
the genome sequences of different organisms. When genes from different 
organisms have very similar nucleotide sequences, it is highly probable 
that both descended from a common ancestral gene. Such genes (and 
their protein producís) are said to be homologous. Now that we have the 
complete genome sequences of many different organisms from all three 
domains of life—archaea, bacteria, and eukaryotes—we can search sys- 
tematically for homologies that span this enormous evolutionary divide. 
By taking stock of the common inheritance of all living things, scientists 
are attempting to trace life's origins back to the earliest ancestral cells. 

Genomes Contain More Than Just Genes 

Although our view of genome sequences tends to be "gene-centric," our 
genomes contain much more than just genes. The vast bulk of our DNA 
does not code for proteins or for functional RNA molecules. Instead, it 
ineludes a mixture of sequences that help regúlate gene activity, plus 
sequences that seem to be dispensable. The large quantity of regulatory 
DNA contained in the genomes of eukaryotic multicellular organisms 
allows for enormous complexity and sophistication in the way different 
genes are brought into action at different times and places. Yet, in the 
end, the basic list of parts—the set of proteins that the cells can make, as 
specified by the DNA—is not much longer than the parts list of an auto- 
mobile, and many of those parts are common not only to all animáis, but 
also to the entire living world. 

That DNA can program the growth, development, and reproduction of 
living cells and complex organisms is truly amazing. In the rest of this 
book, we will tiy to explain what is known about how cells work—by 
examining their component parts, how these parts work together, and 
how the genome of each cell direets the manufacture of the parts the cell 
needs to function and to reproduce. 


• Cells are the fundamental units of life. All present-day cells are 
believed to have evolved from an ancestral cell that existed more 
than 3 billion years ago. 

• All cells are enclosed by a plasma membrane, which separates the 
inside of the cell from its environment. 

• All cells contain DNA as a store of genetic information and use it to 
guide the synthesis of RNA molecules and proteins. 

• Cells in a multicellular organism, though they all contain the same 
DNA, can be veiy different. They turn on different sets of genes 
according to their developmental histoiy and to signáis they receive 
from their environment. 

• Animal and plant cells are typically 5-20 pm in diameter and can be 
seen with a light microscope, which also reveáis some of their inter¬ 
nal components, including the larger organdíes. 

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Cells: The Fundamental Units of Life 

The electrón microscope reveáis even the smallest organdíes, but 
specimens require elabórate preparation and cannot be viewed while 

Specific large molecules can be located in fixed or living cells with a 
fluorescence microscope. 

The simplest of present-day living cells are prokaryotes: although 
they contain DNA, they lack a nucleus and other organdíes and prob- 
ably resemble most closely the ancestral cell. 

Different species of prokaryotes are diverse in their Chemical 
capabilities and inhabit an amazingly wide range of habitats. Two 
fundamental evolutionaiy subdivisions are recognized: bacteria and 

Eukaryotic cells possess a nucleus and other organdíes not found in 
prokaryotes. They probably evolved in a series of stages, including 
the acquisition of mitochondria by engulfment of aerobic bacteria 
and (for plant cells) the acquisition of chloroplasts by engulfment of 
photosynthetic bacteria. 

The nucleus contains the genetic information of the eukaryotic 
organism, stored in DNA molecules. 

The cytoplasm ineludes all of the cell's contents outside the nucleus 
and contains a variety of membrane-enclosed organdíes with spe- 
cialized functions: mitochondria carry out the final oxidation of food 
molecules; in plant cells, chloroplasts perform photosynthesis; the 
endoplasmic reticulum and the Golgi apparatus synthesize complex 
molecules for export from the cell and for insertion in cell mem- 
branes; lysosomes digest large molecules. 

Outside the membrane-enclosed organdíes in the cytoplasm is the 
cytosol, a very concentrated mixture of large and small molecules 
that carry out many essential biochemical processes. 

The cytoskeleton is composed of protein filaments that extend 
throughout the cytoplasm and are responsible for cell shape and 
movement and for the transport of organdíes and other large molec¬ 
ular complexes from one location to another. 

Free-living, single-celled eukaryotic microorganisms are complex 
cells that can swim, mate, hunt, and devour other microorganisms. 
Animáis, plants, and some fungí consist of diverse eukaryotic cell 
types, all derived from a single fertilized egg cell; the number of such 
cells cooperating to form a large multicellular organism such as a 
human runs into thousands of billions. 

Biologists have chosen a small number of model organisms to study 
closely, including the bacterium E. coli, brewer’s yeast, a nematode 
worm, a fly, a small plant, a fish, a mouse, and humans themselves. 
The simplest known cell is a bacterium with about 500 genes, but 
most cells contain significantly more. The human genome has about 
25,000 genes, which is only about twice as many as a fly and six 
times as many as E. coli. 

LibertadDigital \ 2015 

Chapter 1 End-of-Chapter Questions 










fluorescence microscope 




plasma membrane 














model organism 


electrón microscope 



By now you should be familiar with the following cellular 
components. Briefly define what they are and what function 
they provide for cells. 

A. cytosol 

B. cytoplasm 

C. mitochondria 

D. nucleus 

E. chloroplasts 

F. lysosomes 

G. chromosomes 

H. Golgi apparatus 

I. peroxisomes 

J. plasma membrane 

K. endoplasmic reticulum 

L. cytoskeleton 


Which of the following statements are corred? Explain your 

A. The hereditary information of a cell is passed on by its 

B. Baderial DNA is found in the cytosol. 

C. Plants are composed of prokaryotic cells. 

D. All cells of the same organism have the same number of 
chromosomes (with the exception of egg and sperm cells). 

E. The cytosol contains membrane-enclosed organelles, 
such as lysosomes. 

F. The nucleus and mitochondria are surrounded by a 
double membrane. 

G. Protozoans are complex organisms with a set of 
specialized cells that form tissues, such as flagella, 
mouthparts, stinging darts, and leglike appendages. 

H. Lysosomes and peroxisomes are the sites of degradation 
of unwanted materials. 


To get a feeling for the size of cells (and to pradice the use 
of the metric System), consider the following: the human 
brain weighs about 1 kg and contains about 10 12 cells. 
Calcúlate the average size of a brain cell (although we 
know that their sizes vary widely), assuming that each cell is 
entirely filled with water (1 cm 3 of water weighs 1 g). What 
would be the length of one side of this average-sized brain 
cell if it were a simple cube? If the cells were spread out as a 
thin layer that is only a single cell thick, how many pages of 
this book would this layer cover? 


Identify the different organelles indicated with letters in the 
eledron micrograph of a plant cell shown below. Estímate 
the length of the scale bar in the figure. 


There are three major classes of filaments that make up the 
cytoskeleton. What are they, and what are the differences in 

LibertadDigital \ 2015 


CHAPTER 1 Cells: The Fundamental Units of Life 

their functions? Which cytoskeletal filaments would be most 
plentiful in a muscle cell or in an epidermal cell making up 
the outer layer of the skin? Explain your answers. 


Natural selection is such a powerful forcé in evolution 
because cells with even a small proliferation advantage 
quickly outgrow their competitors. To ¡Ilústrate this process, 
consider a cell culture that contains 1 million bacterial cells 
that double every 20 minutes. A single cell in this culture 
acquires a mutation that allows it to divide faster, with a 
generation time of only 15 minutes. Assuming that there is 
an unlimited food supply and no cell death, how long would 
it take before the progeny of the mutated cell became 
predominant in the culture? (Before you go through the 
calculation, make a guess: do you think it would take about 
a day, a week, a month, or a year?) How many cells of either 
type are present in the culture at this time? (The number of 
cells N in the culture at time t is described by the equation 
N = No x 2 t/G , where No is the number of cells at zero time 
and G is the generation time.) 


When bacteria are grown under adverse conditions, i.e., in 
the presence of a poison such as an antibiotic, most cells 
grow and proliferate slowly. But it is not uncommon that the 
growth rate of a bacterial culture kept in the presence of 
the poison is restored after a few days to that observed in 
its absence. Suggest why this may be the case. 


Apply the principie of exponential growth of a culture as 
described in Question 1-13 to the cells in a multicellular 
organism, such as yourself. There are about 10 13 cells 
in your body. Assume that one cell acquires a mutation 
that allows it to divide in an uncontrolled manner (i.e., it 
becomes a cáncer cell). Some cáncer cells can proliferate 
with a generation time of about 24 hours. If none of the 
cáncer cells died, how long would it take before 10 13 cells in 
your body would be cáncer cells? (Use the equation 
N = No x 2 t/G , with t, the time, and G, the length of each 
generation. Hint: 10 13 « 2 43 .) 


Discuss the following statement: "The structure and 
function of a living cell are dictated by the laws of physics 
and chemistry." 


What, if any, are the advantages in being multicellular? 

Draw to scale the outline of two spherical cells, one a 
bacterium with a diameter of 1 [tm, the other an animal cell 
with a diameter of 15 pm. Calcúlate the volume, surface 
area, and surface-to-volume ratio for each cell. How 
would the latter ratio change if you included the internal 
membranes of the cell in the calculation of surface area 
(assume internal membranes have 15 times the area of the 
plasma membrane)? (The volume of a sphere is given by 
47tr 3 /3 and its surface by 4 nr 2 , where r is its radius.) Discuss 
the following hypothesis: "Internal membranes allowed 
bigger cells to evolve." 


What are the arguments that all living cells evolved from 
a common ancestor cell? Imagine the very early days of 
evolution of life on Earth. Would you assume that the 
primordial ancestor cell was the first and only cell to form? 


In Figure 1-25, proteins are blue, nucleic acids are pink, 
lipids are yellow, and polysaccharides are green. Identify 
the major organelles and other important cellular structures 
shown in this slice through a eukaryotic cell. 


Looking at some pond water under the microscope, you 
notice an unfamiliar rod-shaped cell about 200 |tm long. 
Knowing that some exceptional bacteria can be as big 
as this or even bigger, you wonder whether your cell is a 
bacterium or a eukaryote. How will you decide? If it is not a 
eukaryote, how will you discover whether it is a bacterium 
or an archaeon? 

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Chemical Components of Cells 

It is at first sight difficult to accept that living creatures are merely Chemi¬ 
cal Systems. Their incredible diversity of form, their seemingly purposeful 
behavior, and their ability to grow and reproduce all seem to set them 
apart from the world of solids, liquids, and gases that chemistry normally 
describes. Indeed, until the nineteenth century, it was widely believed 
that animáis contained a vital forcé—an "animus"—that was responsible 
for their distinctive properties. 

We now know that there is nothing in living organisms that disobeys 
Chemical or physical laws. However, the chemistry of life is indeed a 
special kind. First, it is based overwhelmingly on carbón compounds, 
the study of which is known as organic chemistry. Second, it depends 
almost exclusively on Chemical reactions that take place in a watery, 
or aqueous, solution and in the relatively narrow range of temperatures 
experienced on Earth. Third, it is enormously complex: even the simplest 
cell is vastly more complicated in its chemistry than any other Chemical 
system known. Fourth, it is dominated and coordinated by collections of 
enormous polymeric molecules —chains of Chemical subunits linked end- 
to-end—whose unique properties enable cells and organisms to grow 
and reproduce and to do all the other things that are characteristic of life. 
Finally, the chemistry of life is tightly regulated: cells deploy a variety of 
mechanisms to make sure that all their Chemical reactions occur at the 
proper place and time. 

Because chemistry lies at the heart of all biology, in this chapter, we briefly 
survey the chemistry of the living cell. We will meet the molecules from 
which cells are made and examine their structures, shapes, and Chemical 
properties. These molecules determine the size, structure, and functions 


LibertadDigital \ 2015 



Chemical Components of Cells 

doud of 

nudeus electrons 

Figure 2-1 An atom consists of a nudeus 
surrounded by an electrón doud. The 

dense, positively charged nudeus contains 
most of the atom's mass. The much lighter 
and negatively charged electrons occupy 
space around the nudeus, as governed 
by the laws of quantum mechanics. The 
electrons are depicted as a continuous 
doud, as there is no way of predicting 
exactly where an electrón ¡s at any given 
¡nstant. The density of shading of the doud 
¡s an ¡ndication ofthe probability that 
electrons will be found there. The diameter 
ofthe electrón doud ranges from about 
0.1 nm (for hydrogen) to about 0.4 nm (for 
atoms of high atomic number). The nudeus 
¡s very much smaller: about 5 x 10 -6 nm for 
carbón, for example. 

Figure 2-2 The number of protons in 
an atom determines its atomic number. 

Schematic representations of an atom of 
carbón and an atom of hydrogen are shown. 
The nudeus of every atom except hydrogen 
consists of both positively charged protons 
and electrically neutral neutrons; the atomic 
weight equals the number of protons plus 
neutrons. The number of electrons in an 
atom is equal to the number of protons, so 
that the atom has no net charge. In contrast 
to Figure 2-1, the electrons are shown 
here as individual particles. The concentric 
b/ac/ccircles represent in a highly schematic 
form the "orbits" (that is, the different 
distributions) ofthe electrons. The neutrons, 
protons, and electrons are in reality minute 
in relation to the atom as a whole; their size 
is greatly exaggerated here. 

of living cells. By understanding how they interact, we can begin to see 
how cells exploit the laws of chemistry and physics to survive, thrive, and 


Matter is made of combinations of elemente —substances such as hydro¬ 
gen or carbón that cannot be broken down or interconverted by Chemical 
means. The smallest particle of an element that still retains its distinctive 
Chemical properties is an atom. The characteristics of substances other 
than puré elements—including the materials from which living cells are 
made—depend on which atoms they contain and the way these atoms 
are linked together in groups to form molecules. To understand living 
organisms, therefore, it is crucial to know how the Chemical bonds that 
hold atoms together in molecules are formed. 

Cells Are Made of Relatively Few Types of Atoms 

Each atom has at its center a dense, positively charged nucleus, which 
is surrounded at some distance by a cloud of negatively charged elec¬ 
trons, held there by electrostatic attraction to the nucleus (Figure 2-1). 
The nucleus consists of two kinds of subatomic particles: protons, which 
are positively charged, and neutrons, which are electrically neutral. The 
number of protons present in an atom's nucleus determines its atomic 
number. An atom of hydrogen has a nucleus composed of a single pro¬ 
tón; so hydrogen, with an atomic number of 1, is the lightest element. 
An atom of carbón has six protons in its nucleus and an atomic number 
of 6 (Figure 2-2). The electric charge carried by each proton is exactly 
equal and opposite to the charge carried by a single electrón. Because 
the whole atom is electrically neutral, the number of negatively charged 
electrons surrounding the nucleus is equal to the number of positively 
charged protons that the nucleus contains; thus the number of electrons 
in an atom also equals the atomic number. All atoms of a given ele¬ 
ment have the same atomic number, and we will see shortly that it is this 
number that dictates each atom's Chemical behavior. 

Neutrons have essentially the same mass as protons. They contribute to 
the structural stability of the nucleus—if there are too many or too few, 
the nucleus may disintegrate by radioactive decay—but they do not alter 
the Chemical properties of the atom. Thus an element can exist in several 
physically distinguishable but chemically identical forms, called iso- 
topes, each having a different number of neutrons but the same number 
of protons. Múltiple isotopes of almost all the elements occur naturally, 

neutrón electrón 

atomic number = 6 
atomic weight = 12 

hydrogen atom 
atomic number = 1 
atomic weight = 1 

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Chemical Bonds 


including some that are unstable—and thus radioactive. For exam- 
ple, while most carbón on Earth exists as the stable isotope carbón 12, 
with six protons and six neutrons, also present are small amounts of an 
unstable isotope, carbón 14, which has six protons and eight neutrons. 
Carbón 14 undergoes radioactive decay at a slow but steady rate, which 
allows archaeologists to estímate the age of organic material. 

The atomic weight of an atom, or the molecular weight of a molecule, 
is its mass relative to that of a hydrogen atom. This is essentially equal to 
the number of protons plus neutrons that the atom or molecule contains, 
because the electrons are so light that they contribute almost nothing to 
the total mass. Thus the major isotope of carbón has an atomic weight 
of 12 and is written as 12 C. The unstable carbón isotope just mentioned 
has an atomic weight of 14 and is written as 14 C. The mass of an atom or 
a molecule is generally specified in daltons, one dalton being an atomic 
mass unit approximately equal to the mass of a hydrogen atom. 

Atoms are so small that it is hard to imagine their size. An individual 
carbón atom is roughly 0.2 nm in diameter, so that it would take about 
5 million of them, laid out in a straight line, to span a millimeter. One 
protón or neutrón weighs approximately 1/(6 x 10 23 ) gram. As hydrogen 
has only one protón—thus an atomic weight of 1—1 gram of hydrogen 
contains 6 x 10 23 atoms. For carbón—which has six protons and six neu¬ 
trons, and an atomic weight of 12—12 grams contain 6 x 10 23 atoms. This 
huge number, called Avogadro's number, allows us to relate everyday 
quantities of Chemicals to numbers of individual atoms or molecules. If 
a substance has a molecular weight of M, M grams of the substance will 
contain 6 x 10 23 molecules. This quantity is called one mole of the sub¬ 
stance (Figure 2-3). The concept of mole is used widely in chemistry as 
a way to represent the number of molecules that are available to particí¬ 
pate in Chemical reactions. 

There are about 90 naturally occurring elements, each differing from the 
others in the number of protons and electrons in its atoms. Living organ- 
isms, however, are made of only a small selection of these elements, four 
of which—carbón (C), hydrogen (H), nitrogen (N), and oxygen (O)—con- 
stitute 96% of an organism's weight. This composition differs markedly 
from that of the nonliving inorganic environment on Earth (Figure 2-4) 
and is evidence of a distinctive type of chemistry. 

A mole is X grams of a substance, 
where X is the molecular weight of the 
substance. A mole will contain 
6 x 10 23 molecules of the substance. 

1 mole of carbón weighs 12 g 
1 mole of glucose weighs 180 g 
1 mole of sodium chloride weighs 58 g 

A one molar solution has a 
concentraron of 1 mole of the substance 
in 1 liter of solution. A 1 M solution of 
glucose, for example, contains 180 g/l, 
and a one millimolar (1 mM) solution 
contains 180 mg/l. 

The standard abbreviation for gram is g; 
the abbreviation for liter is L. 

Figure 2-3 What's a mole? Some sample 
calculations of moles and molar Solutions. 

The Outermost Electrons Determine How Atoms Interact 

To understand how atoms come together to form the molecules that 
make up living organisms, we have to pay special attention to the atoms' 
electrons. Protons and neutrons are welded tightly to one another in an 
atom's nucleus, and they change partners only under extreme condi- 
tions—during radioactive decay, for example, or in the interior of the sun 
or of a nuclear reactor. In living tissues, only the electrons of an atom 
undergo rearrangements. They form the accessible part of the atom and 
specify the rules of chemistry by which atoms combine to form molecules. 
Electrons are in continuous motion around the nucleus, but motions on 
this submicroscopic scale obey different laws from those we are familiar 
with in everyday life. These laws díctate that electrons in an atom can 
exist only in certain discrete regions of movement—roughly speaking, 
in discrete orbits. Moreover, there is a strict limit to the number of elec¬ 
trons that can be accommodated in an orbit of a given type, a so-called 
electrón shell. The electrons closest on average to the positive nucleus 
are attracted most strongly to it and occupy the inner, most tightly bound 
shell. This innermost shell can hold a máximum of two electrons. The 
second shell is farther away from the nucleus, and can hold up to eight 

LibertadDigital \ 2015 


CHAPTER 2 Chemical Components of Cells 

Figure 2-4 The distribution of elements 
in the Earth's crust differs radically from 
that ¡n a living organism. The abundance 
of each element ¡s expressed here as a 
percentage of the total number of atoms 
present ¡n a biological or geological sample, 
¡ncluding water. Thus, for example, more 
than 60% of the atoms ¡n the human body 
are hydrogen atoms, and nearly 30% of the 
atoms in the Earth's crust are Silicon atoms 
(Si). The relative abundance of elements is 
similar in all living things. 


A cup of water, containing exactly 
18 g, or 1 mole, of water, was 
emptied into the Aegean Sea 
3000 years ago. What are the 
chances that the same quantity 
of water, scooped today from the 
Pacific Ocean, would inelude at 
least one of these ancient water 
molecules? Assume perfect mixing 
and an approximate volume for the 
world's oceans of 1.5 billion cubic 
kilometers (1.5 x 10 9 km 3 ). 

| human body 

Earth's crust 


Ljj 1 


H C O N Ca Na P Al Si others 
and and 
Mg K 

electrons. The third Shell can also hold up to eight electrons, which are 
even less tightly bound. The fourth and fifth shells can hold 18 elec¬ 
trons each. Atoms with more than four shells are veiy rare in biological 

The arrangement of electrons in an atom is most stable when all the 
electrons are in the most tightly bound States that are possible for them— 
that is, when they occupy the innermost shells, closest to the nucleus. 
Therefore, with certain exceptions in the larger atoms, the electrons of an 
atom fill the shells in order—the first before the second, the second before 
the third, and so on. An atom whose outermost shell is entirely filled 
with electrons is especially stable and therefore chemically unreactive. 
Examples are helium with 2 electrons (atomic number 2), neón with 2 + 8 
electrons (atomic number 10), and argón with 2 + 8 + 8 electrons (atomic 
number 18); these are all inert gases. Hydrogen, by contrast, has only 
one electrón, which leaves its outermost shell half-filled, so it is highly 
reactive. The atoms found in living organisms all have outermost shells 
that are incompletely filled, and they are therefore able to react with one 
another to form molecules (Figure 2-5). 

Because an incompletely filled electrón shell is less stable than one that 
is completely filled, atoms with incomplete outer shells have a strong 
tendeney to interact with other atoms so as to either gain or lose enough 
electrons to achieve a completed outermost shell. This electrón exchange 
can be achieved either by transferring electrons from one atom to another 
or by sharing electrons between two atoms. These two strategies genér¬ 
ate the two types of Chemical bonds that bind atoms to one another: an 
ionic bond is formed when electrons are donated by one atom to another, 
whereas a covalent bond is formed when two atoms share a pair of elec¬ 
trons (Figure 2-6). 

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Chemical Bonds 


atomic number 

Figure 2-5 An element's Chemical 
reactivity depends on how its outermost 
electrón shell is filled. All of the elements 
commonly found in living organisms have 
outermost shells that are not completely 
filled with electrons (red) and can thus 
particípate in Chemical reactions with other 
atoms. Inert gases (yellow), in contrast, have 
completely filled outermost shells and are 
thus chemically unreactive. 

An H atom, which needs only one more electrón to fill its only shell, gen- 
erally acquires it by sharing—forming one covalent bond with another 
atom. The other most common elements in living cells—C, N, and O, 
which have an incomplete second shell, and P and S, which have an 
incomplete third shell (see Figure 2-5)—generally share electrons and 
achieve a filled outer shell of eight electrons by forming several covalent 
bonds. The number of electrons an atom must acquire or lose (either by 
sharing or by transfer) to attain a filled outer shell determines the number 
of bonds the atom can make. 

Because the State of the outer electrón shell determines the Chemical 
properties of an element, when the elements are listed in order of their 
atomic number we see a periodic recurrence of elements with similar 
properties: an element with, say, an incomplete second shell containing 
one electrón will behave in much the same way as an element that has 
filled its second shell and has an incomplete third shell containing one 
electrón. The metáis, for example, have incomplete outer shells with just 
one or a few electrons, whereas, as we have just seen, the inert gases 
have full outer shells. This arrangement gives rise to the periodic table of 
the elements, outlined in Figure 2-7, which shows elements found in liv¬ 
ing organisms highlighted in color. 

covalent bond 


A carbón atom contains six protons 
and six neutrons. 

A. What are its atomic number and 
atomic weight? 

B. How many electrons does it 

C. How many additional electrons 
must it add to fill its outermost 
shell? How does this affect carbon's 
Chemical behavior? 

D. Carbón with an atomic weight of 
14 is radioactive. How does it differ 
in structure from nonradioactive 
carbón? How does this difference 
affect its chemical behavior? 

Figure 2-6 Atoms can attain a more 
stable arrangement of electrons in their 
outermost shell by interacting with one 
another. A covalent bond is formed when 
electrons are shared between atoms. An 
ionic bond is formed when electrons are 
transferred from one atom to the other. The 
two cases shown represent extremes; often, 
covalent bonds form with a partial transfer 
(unequal sharing of electrons), resulting in a 
polar covalent bond, as we discuss shortly. 

LibertadDigital \ 2015 


CHAPTER 2 Chemical Components of Cells 

Figure 2-7 The chemistry of life is 
predominantly the chemistry of lighter 
elements. When ordered by their atomic 
number ¡nto a periodic table, elements fall 
¡nto groups that show similar properties 
based on the number of electrons each 
element possesses in its outer shell. Atoms 
¡n the same vertical column must gain or 
lose the same number of electrons to attain 
a filled outer shell, and they thus behave 
similarly. Thus, both magnesium (Mg) and 
calcium (Ca) tend to give away the two 
electrons in their outer shells to form ionic 
bonds with atoms such as chlorine (Cl) that 
need extra electrons to complete their outer 

The four elements highlighted in red 
constitute 99% of the total number of atoms 
present in the human body and about 96% 
of our total weight. An additional seven 
elements, highlighted in blue, together 
represent about 0.9% of the total number 
of atoms. Other elements, shown in green, 
are required in trace amounts by humans. 

It remains unclear whether those elements 
shown inyeí/oware essential in humans 

The atomic weights shown here are 
those of the most common ¡sotope of each 

two hydrogen atoms 

bond length: 0.074 ni 

hydrogen molecule 

atomic number 

Covalent Bonds Form by the Sharing of Electrons 

All of the characteristics of a cell depend on the molecules it contains. 
A molecule is a cluster of atoms held together by covalent bonds, in 
which electrons are shared rather than transferred between atoms. The 
shared electrons complete the outer shells of the interacting atoms. In the 
simplest possible molecule—a molecule of hydrogen (H 2 )—two H atoms, 
each with a single electrón, share their electrons, thus fllling their outer- 
most shells. The shared electrons form a cloud of negative charge that 
is densest between the two positively charged nuclei. This electrón den- 
sity helps to hold the nuclei together by opposing the mutual repulsión 
between their positive charges that would otherwise forcé them apart. 
The attractive and repulsive forces are in balance when the nuclei are 
separated by a characteristic distance, called the bond length (Figure 2-8). 
Whereas an H atom can form only a single covalent bond, the other com¬ 
mon atoms that form covalent bonds in cells—O, N, S, and P, as well as 
the all-important C—can form more than one. The outermost shells of 
these atoms, as we have seen, can accommodate up to eight electrons, 
and they form covalent bonds with as many other atoms as necessary to 
reach this number. Oxygen, with six electrons in its outer shell, is most 
stable when it acquires two extra electrons by sharing with other atoms, 
and it therefore forms up to two covalent bonds. Nitrogen, with five outer 
electrons, forms a máximum of three covalent bonds, while carbón, with 
four outer electrons, forms up to four covalent bonds—thus sharing four 
pairs of electrons (see Figure 2-5). 

When one atom forms covalent bonds with several others, these múl¬ 
tiple bonds have deflnite orientations in space relative to one another, 
reflecting the orientations of the orbits of the shared electrons. Cova¬ 
lent bonds between múltiple atoms are therefore characterized by spe- 
cific bond angles, as well as by specific bond lengths and bond energies 
(Figure 2-9). The four covalent bonds that can form around a carbón 

Figure 2-8 The hydrogen molecule is held together by a covalent 
bond. Each hydrogen atom in ¡solation has a single electrón, which 
means that its first (and only) electrón shell is ¡ncompletely filled. By 
coming together, the two atoms are able to share their electrons, 
so that each obtains a completely filled first shell, with the shared 
electrons adopting modified orbits around the two nuclei. The covalent 
bond between the two atoms has a definite length—0.074 nm, which is 
the distance between the two nuclei. If the atoms were closer together, 
the positive nuclei would repel each other; if they were farther apart, 
they would not be able to share electrons as effectively. 

LibertadDigital \ 2015 

Chemical Bonds 


—o — 



propane (CH 3 -CH 2 -CH 3 ) 

Figure 2-9 Covalent bonds are 
characterized by particular geometries. 

(A) The spatial arrangement of the covalent 
bonds that can be formed by oxygen, 
nitrogen, and carbón. (B) Molecules formed 
from these atoms therefore have a precise 
three-dimensional structure defined by the 
bond angles and bond lengths for each 
covalent linkage. A water molecule, for 
example, forms a "V" shape with an angle 
cióse to 109°. 

In these ball-and-stick models, the 
different colored balls represent different 
atoms, and the sticks represent the 
covalent bonds. The colors traditionally 
used to represent the different atoms— 
black for carbón, white for hydrogen, blue 
for nitrogen, and red for oxygen—were 
established by the chemist August Wilhelm 
Hofmann in 1865, when he used a set of 
colored croquet balls to build molecular 
models for a public lecture on "the 
combining power of atoms." 

atom, for example, are arranged as if pointing to the four corners of a reg¬ 
ular tetrahedron. The precise orientation of the covalent bonds around 
carbón produces the three-dimensional geometry of organic molecules. 

There Are Different Types of Covalent Bonds 

Most covalent bonds involve the sharing of two electrons, one donated 
by each participating atom; these are called single bonds. Some covalent 
bonds, however, involve the sharing of more than one pair of electrons. 
Four electrons can be shared, for example, two coming from each par¬ 
ticipating atom; such a bond is called a double bond. Double bonds are 
shorter and stronger than single bonds and have a characteristic effect 
on the three-dimensional geometry of molecules containing them. A sin¬ 
gle covalent bond between two atoms generally allows the rotation of 
one part of a molecule relative to the other around the bond axis. A dou¬ 
ble bond prevenís such rotation, producing a more rigid and less flexible 
arrangement of atoms (Figure 2-10). This restriction has a major influ- 
ence on the three-dimensional shape of many macromolecules. Panel 
2-1 (pp. 66-67) reviews the covalent bonds commonly encountered in 
biological molecules. 

Some molecules contain atoms that share electrons in a way that pro¬ 
duces bonds that are intermedíate in character between single and double 
bonds. The highly stable benzene molecule, for example, is made up of 
a ring of six carbón atoms in which the bonding electrons are evenly 
distributed (although the arrangement is sometimes depicted as an alter- 
nating sequence of single and double bonds, as shown in Panel 2-1). 
When the atoms joined by a single covalent bond belong to different ele- 
ments, the two atoms usually attract the shared electrons to different 
degrees. Covalent bonds in which the electrons are shared unequally in 
this way are known as polar covalent bonds. A polar structure (in the elec- 
trical sense) is one in which the positive charge is concentrated toward 
one end of the molecule (the positive pole) and the negative charge is 
concentrated toward the other end (the negative pole). Oxygen and nitro- 
gen atoms, for example, attract electrons relatively strongly, whereas an 
H atom attracts electrons relatively weakly (because of the relative dif- 
ferences in the positive charges of the nuclei of C, O, N, and H). Thus the 

Figure 2-10 Carbon-carbon double 
bonds are shorter and more rigid than 
carbon-carbon single bonds. (A) The 

ethane molecule, with a single covalent 
bond between the two carbón atoms, shows 
the tetrahedral arrangement of the three 
single covalent bonds between each carbón 
atom and its three attached H atoms. The 
CH3 groups, joined by a covalent C-C 
bond, can roíate relative to one another 
around the bond axis. (B) The double 
bond between the two carbón atoms in a 
molecule of ethene (ethylene) altersthe 
bond geometry of the carbón atoms and 
brings all the atoms into the same plañe; 
the double bond prevenís the rotation of 
one CH2 group relative to the other. 

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CHAPTER 2 Chemical Components of Cells 




Figure 2-11 In polar covalent bonds, the electrons are shared 
unequally. Comparison of electrón distributions ¡n the polar covalent 
bonds ¡n a molecule of water (H2O) and the nonpolar covalent bonds 
in a molecule of oxygen (O2). In H2O, electrons are more strongly 
attracted to the oxygen nucleus than to the H nucleus, as indicated 
by the distributions of the partial negative (8 - ) and partial positive (8 + ) 

covalent bond between O and H, O-H, or between N and H, N-H, is polar 
(Figure 2-11). An atom of C and an atom of H, by contrast, attract elec¬ 
trons more equally. Thus the bond between carbón and hydrogen, C-H, 
is relatively nonpolar. 


Discuss whether the following 
statement is corred: "An ionic 
bond can, in principie, be thought 
of as a very polar covalent bond. 
Polar covalent bonds, then, fall 
somewhere between ionic bonds 
at one end of the spedrum and 
nonpolar covalent bonds at the 
other end." 

Covalent Bonds Vary ¡n Strength 

We have already seen that the covalent bond between two atoms has 
a characteristic length that depends on the atoms involved. A further 
crucial property of any Chemical bond is its strength. Bond strength is 
measured by the amount of energy that must be supplied to break the 
bond, usually expressed in units of either kilocalories per mole (kcal/ 
mole) or kilojoules per mole (kj/mole). A kilocalorie is the amount of 
energy needed to raise the temperature of 1 liter of water by 1 °C. Thus, if 
1 kilocalorie of energy must be supplied to break 6 x 10 23 bonds of a spe- 
cific type (that is, 1 mole of these bonds), then the strength of that bond is 
1 kcal/mole. One kilocalorie is equal to about 4.2 kj, which is the unit of 
energy universally employed by physical scientists and, increasingly, by 
cell biologists as well. 

To get an idea of what bond strengths mean, it is helpful to compare 
them with the average energies of the impacts that molecules continually 
undergo owing to collisions with other molecules in their environment— 
their thermal, or heat, energy. Typical covalent bonds are stronger than 
these thermal energies by a factor of 100, so they are resistant to being 
pulled apart by thermal motions. In living organisms, they are normally 
broken only during specific Chemical reactions that are carefully control- 
led by highly specialized protein catalysts, called enzymes. 

When water is present, covalent bonds are much stronger than ionic 
bonds. In ionic bonds, electrons are transferred rather than shared, as 
we now discuss. 

Ionic Bonds Form by the Gain and Loss of Electrons 

Ionic bonds are usually formed between atoms that can attain a com- 
pletely filled outer shell most easily by donating electrons to—or accepting 
electrons frorn—another atom, rather than by sharing them. For example, 
returning to Figure 2-5, we see that a sodium (Na) atom can achieve a 
filled outer shell by giving up the single electrón in its third shell. By con¬ 
trast, a chlorine (Cl) atom can complete its outer shell by gaining just one 
electrón. Consequently, if a Na atom encounters a Cl atom, an electrón 
can jump from the Na to the Cl, leaving both atoms with filled outer shells. 
The offspring of this marriage between sodium, a soft and intensely reac¬ 
tive metal, and chlorine, a toxic green gas, is table salt (NaCl). 

When an electrón jumps from Na to Cl, both atoms become electri- 
cally charged ions. The Na atom that lost an electrón now has one less 
electrón than it has protons in its nucleus; it therefore has a net single 
positive charge (Na + ). The Cl atom that gained an electrón now has one 
more electrón than it has protons and has a net single negative charge 
(Cl - ). Because of their opposite charges, the Na + and Cl - ions are attracted 

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Chemical Bonds 


sodium atom (Na) chlorine atom (Cl) 


sodium ion (Na*) chloride ion (Cl ) 
sodium chloride (NaCI) 

Figure 2-12 Sodium chloride is held 
together by ionic bonds. (A) An atom 
of sodium (Na) reacts with an atom of 
chlorine (Cl). Electrons of each atom are 
shown ¡n their different shells; electrons 
in the chemically reactive (incompletely 
filled) outermost shells are shown in red. 

The reaction takes place with transfer of a 
single electrón from sodium to chlorine, 
forming two electrically charged atoms, or 
ions, each with complete sets of electrons 
in their outermost shells. The two ions have 
opposite charge and are held together by 
electrostatic attraction. (B) The product of 
the reaction between sodium and chlorine, 
crystalline sodium chloride, contains sodium 
and chloride ions packed closely together 
in a regular array in which the charges are 
exactly balanced. (C) Color photograph of 
crystals of sodium chloride. 

to each other and are thereby held together by an ionic bond (Figure 
2-12A). Ions held together solely by ionic bonds are generally called salts 
rather than molecules. A NaCI crystal contains astronomical numbers of 
Na + and Cl - packed together in a precise three-dimensional array with 
their opposite charges exactly balanced: a crystal only 1 mm across con¬ 
tains about 2 x 10 19 ions of each type (Figure 2-12B and C). 

Because of the favorable interaction between ions and water molecules 
(which are polar), many salts (including NaCI) are highly soluble in water. 
They dissociate into individual ions (such as Na + and Cl - ), each sur- 
rounded by a group of water molecules. Positive ions are called cations, 
and negative ions are called anions. Small inorganic ions such as Na + , Cl - , 
K + , and Ca 2+ play important parts in many biological processes, including 
the electrical activity of nerve cells, as we discuss in Chapter 12. 


What, if anything, is wrong with 
the following statement: "When 
NaCI is dissolved in water, the 
water molecules closest to the ions 
will tend to preferentially orient 
themselves so that their oxygen 
atoms face the sodium ions and 
face away from the chloride ions"? 
Explain your answer. 

Noncovalent Bonds Help Bring Molecules Together 
¡n Cells 

In aqueous solution, ionic bonds are 10-100 times weaker than the cova- 
lent bonds that hold atoms together in molecules. But this weakness has 
its place: much of biology depends on specific but transient interactions 
between one molecule and another. These associations are mediated by 
noncovalent bonds. Although noncovalent bonds are individually quite 
weak, their energies can sum to create an effective forcé between two 

The ionic bonds that hold together the Na + and Cl - ions in a salt ciystal 
(see Figure 2-12) are a form of noncovalent bond called an electrostatic 
attraction. Electrostatic attractions are strongest when the atoms involved 
are fully charged, as are Na + and Cl - . But a weaker electrostatic attraction 
also occurs between molecules that contain polar covalent bonds (see 
Figure 2-11). Polar covalent bonds are thus extremely important in biol¬ 
ogy because they allow molecules to interact through electrical forces. 
Any large molecule with many polar groups will have a pattern of par¬ 
dal positive and negative charges on its surface. When such a molecule 
encounters a second molecule with a complementary set of charges, 
the two will be attracted to each other by electrostatic attraction—even 

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CHAPTER 2 Chemical Components of Cells 

Figure 2-13 A large molecule, such as 
a protein, can bind to another protein 
through complementary charges on the 
surface of each molecule. In the aqueous 
environment of a cell, the many individual 
electrostatic attractions shown would help 
the two proteins stay bound to each other. 

S + 

H hydrogen bond 

5 + 

Figure 2-14 A hydrogen bond can form 
between two water molecules. These 
bonds are largely responsible for water's life- 
sustaining properties—including its ability 
to exist as a liquid at the temperatures 
inside the typical mammalian body. 

though water greatly reduces the attractiveness of these charges in most 
biological settings. When present in large numbers, however, weak non- 
covalent bonds on the surfaces of large molecules can promote strong 
and specific binding (Figure 2-13). 

Hydrogen Bonds Are Important Noncovalent Bonds For 
Many Biological Molecules 

Water accounts for about 70% of a cell's weight, and most intracellular 
reactions occur in an aqueous environment. Life on Earth is thought to 
have begun in the ocean. Thus the properties of water have put a perma- 
nent stamp on the chemistry of living things. 

In each molecule of water (H 2 0), the two H atoms are linked to the O 
atom by covalent bonds. The two H-O bonds are highly polar because 
the O is strongly attractive for electrons, whereas the H is only weakly 
attractive. Consequently, there is an unequal distribution of electrons in 
a water molecule, with a preponderance of positive charge on the two H 
atoms and negative charge on the O (see Figure 2-11). When a positively 
charged región of one water molecule (that is, one of its H atoms) comes 
cióse to a negatively charged región (that is, the O) of a second water 
molecule, the electrical attraction between them can establish a weak 
bond called a hydrogen bond (Figure 2-14). These bonds are much 
weaker than covalent bonds and are easily broken by random thermal 
motions. Thus each bond lasts only an exceedingly short time. But the 
combined effect of many weak bonds is far frorn trivial. Each water mol¬ 
ecule can form hydrogen bonds through its two H atoms to two other 
water molecules, producing a network in which hydrogen bonds are 
being continually broken and formed. It is because of these interlocking 
hydrogen bonds that water at room temperature is a liquid—with a high 
boiling point and high surface tensión—and not a gas. Without hydro¬ 
gen bonds, life as we know it could not exist. The biologically significant 
properties of water are reviewed in Panel 2-2 (pp. 68-69). 

Hydrogen bonds are not limited to water. In general, a hydrogen bond 
can form whenever a positively charged H atom held in one molecule 
by a polar covalent linkage comes cióse to a negatively charged atom— 
typically an oxygen or a nitrogen—belonging to another molecule (see 
Figure 2-14). Hydrogen bonds can also occur between different parts of 
a single large molecule, where they often help the molecule fold into a 
particular shape. The length and strength of hydrogen bonds and of ionic 
bonds are compared to those of covalent bonds in Table 2-1. 

Molecules, such as alcohols, that contain polar bonds and that can form 
hydrogen bonds mix well with water. As mentioned previously, molecules 
carrying positive or negative charges (ions) likewise dissolve readily in 
water. Such molecules are termed hydrophilic, meaning that they are 

Bond type Length* (nm) Strength (kcal/mole) 

in vacuum I in water 



90 [377]** 

90 [377] 

Noncovalent: ¡or 

nic bond 


80 [335] 

3 [12.6] 

Noncovalent: hydrogen bond 


4 [16.7] 

1 [4.2] 

*The bond lengths and strengths Usted are approximate, because the exact valúes 
will depend on the atoms involved. 

**Values in brackets are kj/mole. 1 calorie = 4.184 joules. 

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Chemical Bonds 


"water-loving." A large proportion of the molecules in the aqueous envi- 
ronment of a cell fall into this categoiy, including sugars, DNA, RNA, and 
a majority of proteins. Hydrophobic ("water-fearing") molecules, by con- 
trast, are uncharged and form few or no hydrogen bonds, and they do not 
dissolve in water. 

Hydrocarbons are important hydrophobic cell constituents (see Panel 
2-1, pp. 66-67). In these molecules, the H atoms are covalently linked 
to C atoms by nonpolar bonds. Because the H atoms have almost no net 
positive charge, they cannot form effective hydrogen bonds to other mol¬ 
ecules. This makes the hydrocarbon as a whole hydrophobic—a property 
that is exploited by cells, whose membranes are constructed largely from 
lipid molecules that have long hydrocarbon tails. Because lipids do not 
dissolve in water, they can form the thin membrane barriers that keep 
the aqueous interior of the cell sepárate from the surrounding aqueous 
environment, as we discuss later. 

Some Polar Molecules Form Acids and Bases ¡n Water 

One of the simplest kinds of Chemical reaction, and one that has profound 
significance in cells, takes place when a molecule possessing a highly 
polar covalent bond between a hydrogen and another atom dissolves in 
water. The hydrogen atom in such a bond has given up its electrón almost 
entirely to the companion atom, so it exists as an almost naked positively 
charged hydrogen nucleus—in other words, a proton (H + ). When the polar 
molecule becomes surrounded by water molecules, the proton will be 
attracted to the partial negative charge on the oxygen atom of an adja- 
cent water molecule (see Figure 2-11); this proton can dissociate from 
its original partner and associate instead with the oxygen atom of the 
water molecule, generating a hydronium ion (H 3 0 + ) (Figure 2-15A). The 
reverse reaction also takes place very readily, so one has to imagine an 
equilibrium State in which billions of protons are constantly flitting to and 
fro between one molecule and another in an aqueous solution. 
Substances that release protons when they dissolve in water, thus form- 
ing H 3 0 + , are termed acids. The higher the concentration of H 3 0 + , the 
more acidic the solution. H 3 0 + is present even in puré water, at a concen¬ 
tration of 10 -7 M, as a result of the movement of protons from one water 
molecule to another (Figure 2-15B). By tradition, the H 3 0 + concentration 

O covalent 
J bond 

CH 3 —C / + o 

O —H 




proton moves 

molecule to 
the other 

h 3 o + 




Figure 2-15 Protons move continuously from one water molecule to another 
in aqueous Solutions. (A) The reaction that takes place when a molecule of acetic 
acid dissolves in water. At pH 7, nearly all of the acetic acid molecules are present as 
acétate ions. (B) Water molecules are continually exchanging protons with each other 
to form hydronium and hydroxyl ions. These ions in turn rapidly recombine to form 
water molecules. 

LibertadDigital \ 2015 

50 CHAPTER 2 Chemical Components of Cells 

is usually referred to as the H + concentration, even though most protons 
in an aqueous solution are present as H 3 0 + . To avoid the use of unwieldy 
numbers, the concentration of H+ is expressed using a logarithmic scale 
called the pH scale, as illustrated in Panel 2-2. Puré water has a pH of 
7.0 and is thus neutral—that is, neither acidic (pH < 7) ñor basic (pH > 7). 
Acids are characterized as being strong or weak, depending on how read- 
ily they give up their protons to water. Strong acids, such as hydrochloric 
acid (HCl), lose their protons easily. Acetic acid, on the other hand, is a 
weak acid because it holds on to its proton more tightly when dissolved 
in water. Many of the acids important in the cell—such as molecules 
containing a carboxyl (COOH) group—are weak acids (see Panel 2-2, pp. 
68-69). Their tendency to give up a proton with some reluctance is a use- 
ful characteristic, as it renders the molecules sensitive to changes in pH 
in the cell—a property that can be exploited to regúlate function. 

Because protons can be passed readily to many types of molecules in 
cells, thus altering the molecules' character, the H + concentration inside 
a cell (the pH) must be closely controlled. Acids—especially weak acids— 
will give up their protons more readily if the H + concentration is low and 
will tend to accept them back if the concentration is high. 

The opposite of an acid is a base, which ineludes any molecule that 
accepts a proton when dissolved in water. Just as the defining property 
of an acid is that it raises the concentration of H 3 0 + ions by donating a 
proton to a water molecule, so the defining property of a base is that it 
raises the concentration of hydroxyl (OH - ) ions by removing a proton 
from a water molecule. Thus sodium hydroxide (NaOH) is basic (the term 
alkaline is also used) because it dissociates in aqueous solution to form 
Na + ions and OH~ ions; because it does so readily, NaOH is called a strong 
base. Weak bases—which have a weak tendency to accept a proton from 
water—however, are actually more important in cells. Many biologically 
important weak bases contain an amino (NH 2 ) group, which can genér¬ 
ate OH“ by taking a proton from water: -NH 2 + H 2 0 —► -NH 3 + + OH~ (see 
Panel 2-2, pp. 68-69). 

Because an OH - ion combines with a proton to form a water molecule, 
an increase in the OH - concentration forces a decrease in the H + con¬ 
centration, and vice versa. A puré solution of water thus contains an 
equal concentration (10 -7 M) of both ions, rendering it neutral (pH 7). The 
interior of a cell is also kept cióse to neutral by the presence of buffers: 
mixtures of weak acids and bases that can adjust proton concentrations 
around pH 7 by releasing protons (acids) or taking them up (bases). This 
give-and-take keeps the pH of the cell relatively constant under a variety 
of conditions. 


Having looked at the ways atoms combine to form small molecules and 
how these molecules behave in an aqueous environment, we now exam¬ 
ine the main classes of small molecules found in cells and their biological 
roles. Amazingly, we will see that a few basic categories of molecules, 
formed from a handful of different elements, give rise to all the extraordi- 
nary richness of form and behavior displayed by living things. 

A Cell Is Formed from Carbón Compounds 

If we disregard water, nearly all the molecules in a cell are based on car¬ 
bón. Carbón is outstanding among all the elements in its ability to form 
large molecules; Silicon—an element with the same number of electrons 
in its outer shell—is a poor second. Because a carbón atom is small and 


A. Are there any H 3 0 + ions 
present in puré water at neutral pH 
(i.e., at pH = 7.0)? If so, how are 
they formed? 

B. If they exist, what is the ratio 
of H 3 0 + ions to H 2 0 molecules at 
neutral pH? (Hint: the molecular 
weight of water is 18, and 1 liter 
of water weighs 1 kg.) 

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Small Molecules in Cells 


has four electrons and four vacancies in its outer shell, it can form four 
covalent bonds with other atoms (see Figure 2-9). Most importantly, one 
carbón atom can join to other carbón atoms through highly stable cova¬ 
lent C-C bonds to form chains and rings and henee generate large and 
complex molecules with no obvious upper limit to their size. The small 
and large carbón compounds made by cells are called organic mole¬ 
cules. By contrast, all other molecules, including water, are said to be 

Certain combinations of atoms, such as the methyl (-CH 3 ), hydroxyl 
(-OH), carboxyl (-COOH), carbonyl (-0=0), phosphoryl (-PC> 3 2 “), and 
amino (-NH 2 ) groups, occur repeatedly in organic molecules. Each such 
Chemical group has distinct Chemical and physical properties that influ- 
ence the behavior of the molecule in which the group occurs, including 
whether the molecule tends to gain or lose protons and with which other 
molecules it will interact. Knowing these groups and their Chemical prop¬ 
erties greatly simplifies understanding the chemistry of life. The most 
common Chemical groups and some of their properties are summarized 
in Panel 2-1 (pp. 67-68). 

Cells Contain Four Major Families of Small Organic 

The small organic molecules of the cell are carbón compounds with 
molecular weights in the range 100-1000 that contain up to 30 or so 
carbón atoms. They are usually found free in solution in the cytosol and 
have many different roles. Some are used as monomer subunits to con- 
struct the cell's giant polymeric macromolecules —its proteins, nucleic 
acids, and large polysaccharides. Others serve as energy sources, which 
are broken down and transformed into other small molecules in a maze 
of intracellular metabolic pathways. Many have more than one role in the 
cell—acting, for example, as both a potential subunit for a macromole- 
cule and as an energy source. The small organic molecules are much less 
abundant than the organic macromolecules, accounting for only about 
one-tenth of the total mass of organic matter in a cell. As a rough guess, 
there may be a thousand different kinds of these small organic molecules 
in a typical animal cell. 

All organic molecules are synthesized from—and are broken down 
into—the same set of simple compounds. Both their synthesis and their 
breakdown occur through sequences of simple Chemical changes that 
are limited in variety and follow step-by-step rules. As a consequence, 
the compounds in a cell are chemically related, and most can be clas- 
sified into a small number of distinct families. Broadly speaking, cells 
contain four major families of small organic molecules: the sugars, the 
fatty acids, the amino acids, and the nucleotides (Figure 2-16). Although 
many compounds present in cells do not fit into these categories, these 
four families of small organic molecules, together with the macromol¬ 
ecules made by linking them into long chains, account for a large fraction 
of a cell's mass (Table 2-2). 

small organic building blocks 
of the cell 

larger organic molecules 
of the cell 










Figure 2-16 Sugars, fatty acids, amino 
acids, and nucleotides are the four main 
families of small organic molecules 
in cells. They form the monomeric 
building blocks, or subunits, for larger 
organic molecules, including most of the 
macromolecules and other molecular 
assemblies of the cell. Some, like the sugars 
and the fatty acids, are also energy sources. 

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CHAPTER 2 Chemical Components of Cells 


Percent of 

Approximate number 

total cell 

of types of each class 


of molecule 




Inorganic ions 1 20 

Sugars and precursors 1 250 

Amino acids and precursors 0.4 100 

Nucleotides and precursors 0.4 100 

Fatty acids and precursors 1 50 

Other small molecules 0.2 300 

Phospholipids 2 4* 

Macromolecules (nucleic acids, 24 3000 

proteins, and polysaccharides) 

*There are four classes of phospholipids, each of which exists in many varieties. 

Figure 2-17 The structure of glucose, 
a monosaccharide, can be represented 
in several ways. (A) A structural formula 
in which the atoms are shown as Chemical 
symbols, linked together by solid lines 
representing the covalent bonds. The 
thickened lines are used to indícate the 
plañe of the sugar ring and to show that 
the -H and -OH groups are not in the 
same plañe as the ring. (B) Another kind 
of structural formula that shows the three- 
dimensional structure of glucose in the 
so-called "chair configuraron." 

(C) A ball-and-stick model in which the 
three-dimensional arrangement of the 
atoms in space is indicated. (D) Aspace- 
filling model, which, as well as depicting 
the three-dimensional arrangement of the 
atoms, also gives some idea of their relative 
sizes and of the surface contours of the 
molecule (Movie 2.1). The atoms in (C) and 

(D) are colored as in Figure 2-9: C, black ; H, 
wh/te; O, red. This is the conventional color 
coding for these atoms and will be used 
throughout this book. 

Sugars Are Both Energy Sources and Subunits of 

The simplest sugars —the monosaccharides —are compounds with the 
general formula (CH20) n , where n is usually 3, 4, 5, or 6. Sugars, and the 
larger molecules made from them, are also called carbohydrates because 
of this simple formula. Glucose, for example, has the formula C 6 H 12 O 6 
(Figure 2-17). The formula, however, does not fully define the molecule: 
the same set of carbons, hydrogens, and oxygens can be joined together 
by covalent bonds in a variety of ways, creating structures with different 
shapes. Thus glucose can be converted into a different sugar—mannose 
or galactose—simply by switching the orientations of specific -OH groups 
relative to the rest of the molecule (Panel 2-3, pp. 70-71). Each of these 
sugars, moreover, can exist in either of two forms, called the D-form and 
the L-form, which are mirror images of each other. Sets of molecules with 
the same Chemical formula but different structures are called isomers, and 
mirror-image pairs of such molecules are called optical isomers. Isomers 
are widespread among organic molecules in general, and they play a 

ch 2 oh 

LibertadDigital \ 2015 

Small Molecules in Cells 


major part in generating the enormous variety of sugars. A more com¬ 
plete outline of sugar structures and chemistry is presented in Panel 2-3. 
Monosaccharides can be linked by covalent bonds—called glycosidic 
bonds—to form larger carbohydrates. Two monosaccharides linked 
together make a disaccharide, such as sucrose, which is composed of 
a glucose and a fructose unit. Larger sugar polymers range from the oli- 
gosaccharídes (trisaccharides, tetrasaccharides, and so on) up to giant 
polysaccharides, which can contain thousands of monosaccharide units. 
In most cases, the prefix oligo- is used to refer to molecules made of a 
small number of monomers, typically 2 to 10 in the case of oligosac- 
charides. Polymers, in contrast, can contain hundreds or thousands of 

The way sugars are linked together illustrates some common features of 
biochemical bond formation. A bond is formed between an -OH group 
on one sugar and an -OH group on another by a condensation reaction, 
in which a molecule of water is expelled as the bond is formed. The sub¬ 
units in other biological polymers, including nucleic acids and proteins, 
are also linked by condensation reactions in which water is expelled. The 
bonds created by all of these condensation reactions can be broken by 
the reverse process of hydrolysis, in which a molecule of water is con- 
sumed (Figure 2-18). 

Because each monosaccharide has several free hydroxyl groups that can 
form a link to another monosaccharide (or to some other compound), 
sugar polymers can be branched, and the number of possible polysac- 
charide structures is extremely large. For this reason, it is much more 
difficult to determine the arrangement of sugars in a complex polysac- 
charide than to determine the nucleotide sequence of a DNA molecule or 
the amino acid sequence of a protein, in which each unit is joined to the 
next in exactly the same way. 


Figure 2-18 Two monosaccharides can 
be linked by a covalent glycosidic bond 
to form a disaccharide. This reaction 
belongs to a general category of reactions 
termed condensation reactions, in which 
two molecules join together as a result of 
the loss of a water molecule. The reverse 
reaction (in which water is added) is termed 

The monosaccharide glucose has a central role as an energy source for 
cells. It is broken down to smaller molecules in a series of reactions, 
releasing energy that the cell can hamess to do useful work, as we 
explain in Chapter 13. Cells use simple polysaccharides composed only 
of glucose units—principally glycogen in animáis and starch in plants—as 
long-term stores of glucose, held in reserve for energy production. 

Sugars do not function exclusively in the production and storage of 
energy. They are also used, for example, to make mechanical supports. 
The most abundant organic molecule on Earth—the cellulose that forms 
plant cell walls—is a polysaccharide of glucose. Another extraordinarily 
abundant organic substance, the chitin of insect exoskeletons and fungal 
cell walls, is also a polysaccharide—in this case, a linear polymer of a 
sugar derivative called N-acetylglucosamine (see Panel 2-3, pp. 70-71). 
Other polysaccharides, which tend to be slippery when wet, are the main 
components of slime, mucus, and gristle. 

Smaller oligosaccharides can be covalently linked to proteins to form 
glycoproteins, or to lipids to form glycolipids (Panel 2-4, pp. 72-73), which 
are both found in cell membranes. The sugar side chains attached to 
glycoproteins and glycolipids in the plasma membrane are thought to 
help protect the cell surface and often help cells adhere to one another. 
Differences in the types of cell-surface sugars form the molecular basis 
for different human blood groups. 

Fatty Acid Chains Are Components of Cell Membranes 

A fatty acid molecule, such as palmitic acid, has two chemically distinct 
regions. One is a long hydrocarbon chain, which is hydrophobic and 
not very reactive chemically. The other is a carboxyl (-COOH) group, 

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CHAPTER 2 Chemical Components of Cells 

Figure 2-19 Fatty acids have both 
hydrophobic and hydrophilic components. 

The hydrophobic hydrocarbon chain ¡s 
attached to a hydrophilic carboxylic acid 
group. Different fatty acids have different 
hydrocarbon tails. Palmitic acid is shown 
here. (A) Structural formula, showing the 
carboxylic acid head group ¡n its ionized 
form, as it exists in water at pH 7, (B) Ball- 
and-stick model. (C) Space-filling model 
(Movie 2.2). 

saturated unsaturated 

fatty acid tails fatty acid tails 
(A) (B) 

Figure 2-20 The properties of fats 
depend on the length and saturation of 
the fatty acid chains they carry. Fatty acids 
are stored in the cytoplasm of many cells 
in the form of droplets of triacylglycerol 
molecules made of three fatty acid chains 
joined to a glycerol molecule. (A) Saturated 
fats are found in meat and dairy producís. 

(B) Plant oils, such as corn oil, contain 
unsaturated fatty acids, which may be 
monounsaturated (containing one double 
bond) or polyunsaturated (containing 
múltiple double bonds); this is why plant oils 
are liquid at room temperature. Aithough 
fats are essential in the diet, saturated fats 
are not: they raise the concentraron of 
cholesterol in the blood, which tends to 
clog the arteries, increasing the risk of heart 
attacks and strokes. 

which behaves as an acid (carboxylic acid): in an aqueous solution, 
it is ionized (-COO - ), extremely hydrophilic, and chemically reactive 
(Figure 2-19). Almost all the fatty acid molecules in a cell are covalently 
linked to other molecules by their carboxylic acid group (see Panel 2-4, 
pp. 72-73). Molecules—such as fatty acids—that possess both hydropho¬ 
bic and hydrophilic regions are termed amphipathic. 

The hydrocarbon tail of palmitic acid is saturated: it has no double bonds 
between its carbón atoms and contains the máximum possible number 
of hydrogens. Some other fatty acids, such as oleic acid, have unsatu¬ 
rated tails, with one or more double bonds along their length. The double 
bonds create kinks in the hydrocarbon tails, interfering with their abil- 
ity to pack together, and it is the absence or presence of these double 
bonds that accounts for the difference between hard (saturated) and soft 
(polyunsaturated) margarine. Fatty acid tails are also found in cell mem- 
branes, where the tightness of their packing affects the fluidity of the 
membrane. The many different fatty acids found in cells differ only in the 
length of their hydrocarbon chains and in the number and position of the 
carbon-carbon double bonds (see Panel 2-4). 

Fatty acids serve as a concentrated food reserve in cells: they can be bro- 
ken down to produce about six times as much usable energy, weight for 
weight, as glucose. Fatty acids are stored in the cytoplasm of many cells 
in the form of fat droplets composed of triacylglycerol molecules—com- 
pounds made of three fatty acid chains covalently joined to a glycerol 
molecule (Figure 2-20, and see Panel 2-4). Triacylglycerols are the ani¬ 
mal fats found in meat, butter, and cream, and the plant oils such as 
corn oil and olive oil. When a cell needs energy, the fatty acid chains 
can be released from triacylglycerols and broken down into two-carbon 
units. These two-carbon units are identical to those derived from the 
breakdown of glucose, and they enter the same energy-yielding reaction 
pathways, as described in Chapter 13. 

Fatty acids and their derivatives, including triacylglycerols, are examples 
of lipids. Lipids are loosely defined as molecules that are insoluble in 
water but soluble in fat and organic solvents such as benzene. They typi- 
cally contain long hydrocarbon chains, as in the fatty acids, or múltiple 
linked aromatic rings, as in the steroids (see Panel 2-4). 

The most unique function of fatty acids is in the formation of the lipid 
bilayer, which is the basis for all cell membranes. These thin sheets, 

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Small Molecules in Cells 


Figure 2-21 Phospholipids can aggregate 
to form cell membranes. Phospholipids 
are composed of two hydrophobic fatty 
acid tails joined to a hydrophilic head. In an 
aqueous environment, the hydrophobic tails 
pack together to exelude water, forming a 
lipid bilayer, with the hydrophilic heads of 
the phospholipid molecules on the outside, 
facing the aqueous environment, and the 
hydrophobic tails on the inside. 

which endose all cells and surround their intemal organelles, are com¬ 
posed largely of phospholipids (Figure 2-21). 

Like triacylglycerols, most phospholipids are constructed mainly from fatty 
acids and glycerol. In these phospholipids, however, the glycerol is joined 
to two fatty acid chains, rather than to three as in triacylglycerols. The 
remaining -OH group on the glycerol is linked to a hydrophilic phosphate 
group, which in turn is attached to a small hydrophilic compound such 
as choline (see Panel 2-4, pp. 72-73). With their two hydrophobic fatty 
acid tails and a hydrophilic, phosphate-containing head, phospholipids 
are strongly amphipathic. This characteristic amphipathic composition 
and shape gives them different physical and Chemical properties from 
triacylglycerols, which are predominantly hydrophobic. In addition to 
phospholipids, cell membranes contain differing amounts of other lip- 
ids, including glycolipids, which contain one or more sugars instead of a 
phosphate group. 

Thanks to their amphipathic nature, phospholipids readily form mem¬ 
branes in water. These lipids will spread over the surface of water to 
form a monolayer, with their hydrophobic tails facing the air and their 
hydrophilic heads in contact with the water. Two such molecular layers 
can readily combine tail-to-tail in water to form the phospholipid sand¬ 
wich that is the lipid bilayer (see Chapter 11). 

Armiño Acids Are the Subunits of Proteins 

Amino acids are small organic molecules with one defining property: 
they all possess a carboxylic acid group and an amino group, both linked 
to their a-carbon atom (Figure 2-22). Each amino acid also has a side 
chain attached to its a-carbon. The identity of this side chain is what dis- 
tinguishes one amino acid from another. 

group group 

nonionized form ionized form 

(A) (B) (0 

Figure 2-22 All amino acids have an 
amino group, a carboxyl group, and a 
side chain (R) attached to their a-carbon 
atom. In the cell, where the pH is cióse to 
7, free amlno acids exlst ¡n their ionized 
form; but, when they are incorporated into 
a polypeptide chain, the charges on their 
amino and carboxyl groups disappear. 

(A) The amino acid shown is alanine, one 
of the simplest amino acids, which has a 
methyl group (CH3) as its side chain. 

(B) A ball-and-stick model and (C) a space- 
filling model of alanine. In (B) and (C), the 
N atom is blue. 

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CHAPTER 2 Chemical Components of Cells 

N-terminus of 
polypeptide chain 

C-terminus of 
polypeptide chain 


Why do you suppose only L-amino 
acids and not a random mixture of 
the l- and D-forms of each amino 
acid are used to make proteins? 

Figure 2-23 Amino acids ¡n a protein are held together by peptide 
bonds. The four amino acids shown are linked together by three 
peptide bonds, one of which ¡s highlighted ¡n yellow. One of the 
amino acids, glutamic acid, is shaded in gray. The amino acid side 
chains are shown in red. The two ends of a polypeptide chain are 
chemically distinct. One end, the N-terminus, is capped by an amino 
group, and the other, the C-terminus, ends in a carboxyl group. The 
sequence of amino acids in a protein is abbreviated using either a 
three-letter or a one-letter code, and the sequence is always read from 
the N-terminus (see Panel 2-5, pp. 74-75). In the example given, the 
sequence is Phe-Ser-Glu-Lys (or FSEK). 

Cells use amino acids to build proteins —polymers made of amino acids, 
which are joined head-to-tail in a long chain that folds up into a three- 
dimensional structure that is unique to each type of protein. The covalent 
bond between two adjacent amino acids in a protein chain is called a 
peptide bond■ the chain of amino acids is also known as a polypeptide. 
Peptide bonds are formed by condensation reactions that link one amino 
acid to the next. Regardless of the speciflc amino acids from which it is 
made, the polypeptide always has an amino (NH 2 ) group at one end—its 
N-terminus —and a carboxyl (COOH) group at its other end—its C-terminus 
(Figure 2-23). This difference in the two ends gives a polypeptide a defi- 
nite directionality—a structural (as opposed to electrical) polarity. 

Twenty types of amino acids are commonly found in proteins, each with a 
different side chain attached to the a-carbon atom (Panel 2-5, pp. 74-75). 
The same 20 amino acids are found in all proteins, whether they hail 
from bacteria, plants, or animáis. How this precise set of 20 amino acids 
carne to be chosen is one of the mysteries surrounding the evolution of 
life; there is no obvious Chemical reason why other amino acids could 
not have served just as well. But once the selection had been locked into 
place, it could not be changed, as too much chemistiy had evolved to 
exploit it. Switching the types of amino acids used by cells would require 
a living creature to retool its entire metabolism to cope with the new 
building blocks. 

Like sugars, all amino acids (except glycine) exist as optical isomers in d- 
and L-forms (see Panel 2-5). But only L-forms are ever found in proteins 
(although D-amino acids occur as part of bacterial cell walls and in some 
antibiotics, and D-serine is used as a signal molecule in the brain). The 
origin of this exclusive use of L-amino acids to make proteins is another 
evolutionary mystery. 

The Chemical versatility that the 20 standard amino acids provide is vitally 
important to the function of proteins. Five of the 20 amino acids—includ- 
ing lysine and glutamic acid, shown in Figure 2-23—have side chains that 
can form ions in solution and can therefore carry a charge. The others 
are uncharged. Some amino acids are polar and hydrophilic, and some 
are nonpolar and hydrophobic (see Panel 2-5). As we discuss in Chapter 
4, the collective properties of the amino acid side chains underlie all the 
diverse and sophisticated functions of proteins. 

Nucleotides Are the Subunits of DNA and RNA 

DNA and RNA are built from subunits called nucleotides. Nucleosides 
are made of a nitrogen-containing ring compound linked to a five-carbon 
sugar, which can be either ribose or deoxyribose (Panel 2-6, pp. 76-77). 
Nucleotides are nucleosides that contain one or more phosphate groups 
attached to the sugar, and they come in two main forms: those containing 
ribose are known as ribonucleotides, and those containing deoxyribose 
are known as deoxyribonucleotídes. 

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Small Molecules in Cells 


Figure 2-24 Adenosine triphosphate 
(ATP) ¡s a crucially important energy 
carrier in cells. (A) Structural formula, in 
which the three phosphate groups are 
shaded in yellow. (B) Ball-and-stick model 
(Movie 2.3). In (B), the P atoms are yellow. 

The nitrogen-containing rings of all these molecules are generally referred 
to as bases for historical reasons: under acidic conditions, they can each 
bind an H + (proton) and thereby increase the concentration of OH - ions 
in aqueous solution. There is a strong family resemblance between the 
different nucleotide bases. Cytosine (C), thymine (T), and uracil (U) are 
called pyrimidines, because they all derive from a six-membered pyrimi- 
dine ring; guanine (G) and adenine (A) are purínes, which bear a second, 
flve-membered ring fused to the six-membered ring. Each nucleotide is 
named after the base it contains (see Panel 2-6, pp. 76-77). 

Nucleotides can act as short-term carriers of Chemical energy. Above 
all others, the ribonucleotide adenosine triphosphate, or ATP (Figure 
2-24), participates in the transfer of energy in hundreds of metabolic 
reactions. ATP is formed through reactions that are driven by the energy 
released by the breakdown of foodstuffs. Its three phosphates are linked 
in series by two phosphoanhydride bonds (see Panel 2-6). Rupture of these 
phosphate bonds releases large amounts of useful energy. The terminal 
phosphate group in particular is frequently split off by hydrolysis (Figure 
2-25) . In many situations, transfer of this phosphate to other molecules 
releases energy that drives energy-requiring biosynthetic reactions. Other 
nucleotide derivatives serve as carriers for the transfer of other Chemical 
groups. All of this is described in Chapter 3. 

Nucleotides also have a fundamental role in the storage and retrieval of 
biological information. They serve as building blocks for the construction 

Figure 2-25 ATP is synthesized from ADP 
and ¡norganic phosphate, and it releases 
energy when it is hydrolyzed back to 
ADP and inorganic phosphate. The energy 
required for ATP synthesis is derived from 
either the energy-yielding oxidation of 
foodstuffs (in animal cells, fungí, and some 
bacteria) or the capture of light (in plant 
cells and some bacteria). The hydrolysis 
of ATP provides the energy to drive many 
processes inside cells. Together, the two 
reactions shown form the ATP cycle. 

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CHAPTER 2 Chemical Components of Cells 

5 end 

"O— P«¡ 




J-g#?í=U NH 2 

T «dh" ■ 




"O— p=o o 

¿ H 3C y^ NH t 

CH 2 o 



"O— priÉJ| NH 2 

? ^ ■ 

5'CH 2 o 


Figure 2-26 A short length of one chain of a deoxyribonucleic 
acid (DNA) molecule shows the covalent phosphodiester bonds 
linking four consecutive nucleotides. Because the bonds link specific 
carbón atoms in the sugar ring—known as the 5' and 3' atoms—one 
end of a polynucleotide chain, the 5' end, has a free phosphate group 
and the other, the 3' end, has a free hydroxyl group. One of the 
nucleotides, thymine (T), ¡s shaded in gray, and one phosphodiester 
bond is highlighted in yellow. The linear sequence of nucleotides in a 
polynucleotide chain is commonly abbreviated by a one-letter code, 
and the sequence is always read from the 5' end. In the example 
¡llustrated, the sequence is GATC. 

of nucleic acids —long polymers in which nucleotide subunits are linked 
by the formation of covalent phosphodiester bonds between the phos¬ 
phate group attached to the sugar of one nucleotide and a hydroxyl 
group on the sugar of the next nucleotide (Figure 2-26). Nucleic acid 
chains are synthesized from energy-rich nucleoside triphosphates by 
a condensation reaction that releases inorganic pyrophosphate during 
phosphodiester bond formation (see Panel 2-6, pp. 76-77). 

There are two main types of nucleic acids, which differ in the type of 
sugar contained in their sugar-phosphate backbone. Those based on the 
sugar ribose are known as ribonucleic acids, or RNA, and contain the 
bases A, G, C, and U. Those based on deoxyribose (in which the hydroxyl 
at the 2' position of the ribose carbón ring is replaced by a hydrogen) are 
known as deoxyribonucleic acids, or DNA, and contain the bases A, G, 
C, and T (T is chemically similar to the U in RNA; see Panel 2-6). RNA usu- 
ally occurs in cells in the form of a single-stranded polynucleotide chain, 
but DNA is virtually always in the form of a double-stranded molecule: 
the DNA double helix is composed of two polynucleotide chains that run 
in opposite directions and are held together by hydrogen bonds between 
the bases of the two chains (Panel 2-7, pp. 78-79). 

The linear sequence of nucleotides in a DNA or an RNA molecule encodes 
genetic information. The two nucleic acids, however, have different roles 
in the cell. DNA, with its more stable, hydrogen-bonded hélices, acts as 
a long-term repository for hereditary information, while single-stranded 
RNA is usually a more transient carrier of molecular instructions. The 
ability of the bases in different nucleic acid molecules to recognize and 
pair with each other by hydrogen-bonding (called base-pairing )—G with 
C, and A with either T or U—underlies all of heredity and evolution, as 
explained in Chapter 5. 


On the basis of weight, macromolecules are by far the most abundant of 
the organic molecules in a living cell (Figure 2-27). They are the principal 
building blocks from which a cell is constructed and also the components 
that confer the most distinctive properties on living things. Intermedíate 
in size and complexity between small organic molecules and organdíes, 
macromolecules are constructed simply by covalently linking small 
organic monomers, or subunits, into long chains, or polymers (Figure 
2-28 and How We Know, pp. 60-61). Yet they have many unexpected 
properties that could not have been predicted from their simple constitu- 
ents. For example, it took a long time to determine that the nucleic acids 
DNA and RNA store and transmit hereditary information (see How We 
Know, pp. 174-176). 

Proteins are especially versatile and perform thousands of distinct func- 
tions in cells. Many proteins act as enzymes that catalyze the Chemical 

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Macromolecules in Cells 


Figure 2-27 Macromolecules are abundant in cells. The approximate composition 
(by mass) of a bacterial cell is shown. The composition of an animal cell ¡s similar. 





nudeic acid 

Figure 2-28 Polysaccharides, proteins, 
and nucleic acids are made from 
monomeric subunits. Each macromolecule 
is a polymerformed from small molecules 
(called monomers or subunits) that are 
linked together by covalent bonds. 

reactions that take place in cells. For example, an enzyme in plants, called 
ribulose bisphosphate carboxylase, converts C0 2 to sugars, thereby creat- 
ing most of the organic matter used by the rest of the living world. Other 
proteins are used to build structural components: tubulin, for example, 
self-assembles to make the cell's long, stiff microtubules (see Figure 

1- 27B), and histone proteins assemble into spool-like structures that 
help wrap up the cell's DNA in chromosomes. Yet other proteins, such 
as myosin, act as molecular motors to produce forcé and movement. We 
examine the molecular basis for many of these wide-ranging functions in 
later chapters. Here, we consider some of the general principies of mac- 
romolecular chemistiy that make all of these activities possible. 

Each Macromolecule Contains a Specific Sequence of 

Although the Chemical reactions for adding subunits to each polymer 
are different in detail for proteins, nucleic acids, and polysaccharides, 
they share important features. Each polymer grows by the addition of a 
monomer onto one end of the polymer chain via a condensation reac- 
tion, in which a molecule of water is lost with each subunit added (Figure 

2- 29) . In all cases, the reactions are catalyzed by specific enzymes, which 
ensure that only the appropriate monomer is incorporated. 

The stepwise polymerization of monomers into a long chain is a simple 
way to manufacture a large, complex molecule, because the subunits are 
added by the same reaction performed over and over again by the same 
set of enzymes. In a sense, the process resembles the repetitive opera- 
tion of a machine in a factory—with some important differences. First, 
apart from some of the polysaccharides, most macromolecules are made 
from a set of monomers that are slightly different from one another; for 
example, proteins are constructed from 20 different amino acids (see 
Panel 2-5, pp. 74-75). Second, and most important, the polymer chain is 
not assembled at random from these subunits; instead the subunits are 
added in a particular order, or sequence. 

The biological functions of proteins, nucleic acids, and many polysac¬ 
charides are absolutely dependent on the particular sequence of subunits 
in the linear chains. By vaiying the sequence of subunits, the cell can 
make an enormous diversity of the polymeric molecules. Thus, for a pro¬ 
tein chain 200 amino acids long, there are 20 200 possible combinations 
(20 x 20 x 20 x 20... multiplied 200 times), while for a DNA molecule 


What is meant by "polarity" of a 
polypeptide chain and by "polarity" 
of a chemical bond? How do the 
meanings differ? 

subunit growing polymer 


Figure 2-29 Macromolecules are formed 
by adding subunits to one end. In a 

condensation reaction, a molecule of water 
is lost with the addition of each monomer to 
one end of the growing chain. The reverse 
reaction—the breakdown of the polymer— 
occurs by the addition of water (hydrolysis). 
See also Figure 2-18. 

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The idea that proteins, polysaccharides, and nucleic 
acids are large molecules that are constructed from 
smaller subunits, linked one after another into long 
molecular chains, may seem fairly obvious today. But 
this was not always the case. In the early part of the 
twentieth centuiy, few scientists believed in the exis- 
tence of such biological polymers built from repeating 
units held together by covalent bonds. The notion that 
such "frighteningly large" macromolecules could be 
assembled from simple building blocks was considered 
"downright shocking" by chemists of the day. Instead, 
they thought that proteins and other seemingly large 
organic molecules were simply heterogeneous aggre- 
gates of small organic molecules held together by weak 
"association forces" (Figure 2-30). 

The flrst hint that proteins and other organic polymers 
are large molecules carne from observing their behav- 
ior in solution. At the time, scientists were working with 
various proteins and carbohydrates derived from food- 
stuffs and other organic materials—albumin from egg 
whites, casein from milk, collagen from gelatin, and cel- 
lulose from wood. Their Chemical compositions seemed 
simple enough: like other organic molecules, they con- 
tained carbón, hydrogen, oxygen, and, in the case of 
proteins, nitrogen. But they behaved oddly in solution, 
showing, for example, an inability to pass through a fine 

Why these molecules misbehaved in solution was a 
puzzle. Were they really giant molecules, composed of 
an unusual number of covalently linked atoms? Or were 
they more like a colloidal suspensión of particles—a 
big, sticky hodgepodge of small organic molecules that 
associate only loosely? 


Figure 2-30 What might an organic macromolecule look 
like? Chemists ¡n the early part of the twentieth century debated 
whether proteins, polysaccharides, and other apparently large 
organic molecules were (A) discrete particles made of an 
unusually large number of covalently linked atoms or (B) a loose 
aggregation of heterogeneous small organic molecules held 
together by weak forces. 

One way to distinguish between the two possibilities was 
to determine the actual size of one of these molecules. 
If a protein such as albumin were made of molecules all 
identical in size, that would support the existence of true 
macromolecules. Conversely, if albumin were instead a 
miscellaneous conglomeration of small organic mole¬ 
cules, these should show a whole range of molecular 
sizes in solution. 

Unfortunately, the techniques available to scientists in 
the early 1900s were not ideal for measuring the sizes of 
such large molecules. Some chemists estimated a pro- 
tein's size by determining how much it would lower a 
solution's freezing point; others measured the osmotic 
pressure of protein Solutions. These methods were sus¬ 
ceptible to experimental error and gave variable results. 
Different techniques, for example, suggested that cel- 
lulose was anywhere from 6000 to 103,000 daltons in 
mass (where 1 dalton is approximately equal to the 
mass of a hydrogen atom). Such results helped to fuel 
the hypothesis that carbohydrates and proteins were 
loose aggregates of small molecules rather than true 

Many scientists simply had trouble believing that 
molecules heavier than about 4000 daltons—the larg- 
est compound that had been synthesized by organic 
chemists—could exist at all. Take hemoglobin, the oxy- 
gen-carrying protein in red blood cells. Researchers tried 
to estímate its size by breaking it down into its Chemical 
components. In addition to carbón, hydrogen, nitro- 
gen, and oxygen, hemoglobin contains a small amount 
of iron. Working out the percentages, it appeared that 
hemoglobin had one atom of iron for every 712 atoms 
of carbón—and a mínimum weight of 16,700 daltons. 
Could a molecule with hundreds of carbón atoms in one 
long chain remain intact in a cell and perform specific 
functions? Emil Fischer, the organic chemist who deter- 
mined that the amino acids in proteins are linked by 
peptide bonds, thought that a polypeptide chain could 
grow no longer than about 30 or 40 amino acids. As 
for hemoglobin, with its purported 700 carbón atoms, 
the existence of molecular chains of such "truly fantas- 
tic lengths" was deemed "very improbable" by leading 

Deflnitive resolution of the debate had to await the devel- 
opment of new techniques. Convincing evidence that 
proteins are macromolecules carne from studies using 
the ultracentrifuge—a device that uses centrifugal forcé 
to sepárate molecules according to their size (see Panel 
4-3, pp. 164-165). Theodor Svedberg, who designed the 
machine in 1925, performed the first studies. If a pro¬ 
tein were really an aggregate of smaller molecules, he 

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Macromolecules in Cells 


reasoned, it would appear as a smear of molecules of 
different sizes when sedimented in an ultracentrifuge. 
Using hemoglobin as his test protein, Svedberg found 
that the centrifuged sample revealed a single, sharp 
band with a molecular weight of 68,000 daltons. His 
results strongly supported the theoiy that proteins are 
true macromolecules (Figure 2-31). 

Additional evidence continued to accumulate through- 
out the 1930s, as other researchers began to prepare 
ciystals of puré protein that could be studied by X-ray dif- 
fraction. Only molecules with a uniform size and shape 
can form highly ordered ciystals and diffract X-rays in 
such a way that their three-dimensional structure can be 
determined, as we discuss in Chapter 4. A heterogene- 
ous suspensión could not be studied in this way. 

We now take it for granted that large macromolecules 
cariy out many of the most important activities in living 
cells. But chemists once viewed the existence of such 
polymers with the same sort of skepticism that a zoolo- 
gist might show on being told that "In Africa, there are 
elephants that are 100 meters long and 20 meters tall." 
It took decades for researchers to master the techniques 
required to convince everyone that molecules ten times 
larger than anything they had ever encountered were a 
comerstone of biology. As we shall see throughout this 
book, such a labored pathway to discoveiy is not un- 
usual, and progress in Science is often driven by 
advances in technology. 

the sample is loaded as a 
narrow band at the top of 
the tu be 








aggregates would 
sedlment to 
produce a 

sediments as ¡ 
single band 



Figure 2-31 The ultracentrifuge helped to settle the debate about the nature of macromolecules. In the ultracentrifuge, 
centrifuga! forces exceeding 500,000 times the forcé of gravity can be used to sepárate proteins or other large molecules. (A) In a 
modern ultracentrifuge, samples are loaded in a thin layer on top of a gradient of sucrose solution formed in a tube. The tube is placed 
in a metal rotor that is rotated at high speed. Molecules of different sizes sediment at different rates, and these molecules will therefore 
move as distinct bands in the sample tube. If hemoglobin were a loose aggregate of heterogeneous peptides, it would show a broad 
smear of sizes after centrifugation (top tube). Instead, it appears as a sharp band with a molecular weight of 68,000 daltons (bottom 
tube). Although the ultracentrifuge is now a standard, almost mundane, fixture in most biochemistry laboratories, its construction was 
a huge technological challenge. The centrifuge rotor must be capable of spinning centrifuge tubes at high speeds for many hours 
at constanttemperature and with high stability; otherwise convection occurs in the sedimenting solution and ruins the experiment. 

In 1926, Svedberg won the Nobel Prize in Chemistry for his ultracentrifuge design and its application to chemistry. (B) In his actual 
experiment, Svedberg filled a special tube in the centrifuge with a homogeneous solution of hemoglobin; by shining light through the 
tube, he then carefully monitored the moving boundary between the sedimenting protein molecules and the clear aqueous solution left 
behind (so-called boundary sedimentation). The more recently developed method shown in (A) is a form of band sedimentation. 

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62 CHAPTER 2 Chemical Components of Cells 

10,000 nucleotides long (small by DNA standards), with its four differ- 
ent nucleotides, there are 4 1 °' 000 different possibilities—an unimaginably 
large number. Thus the machinery of polym eriza ti on must be subject to a 
sensitive control that allows it to specify exactly which subunit should be 
added next to the growing polymer end. We discuss the mechanisms that 
specify the sequence of subunits in DNA, RNA, and protein molecules in 
Chapters 6 and 7. 

Noncovalent Bonds Specify the Precise Shape 
of a Macromolecule 

Most of the single covalent bonds that link together the subunits in a mac¬ 
romolecule allow rotation of the atoms they join; thus the polymer chain 
has great flexibility. In principie, this allows a single-chain macromol¬ 
ecule to adopt an almost unlimited number of shapes, or conformations, 
as the polymer chain writhes and rotates under the influence of random 
thermal energy. However, the shapes of most biological macromolecules 
are highly constrained because of weaker, noncovalent bonds that form 
between different parís of the molecule. In many cases, these weaker 
interactions ensure that the polymer chain preferentially adopts one par¬ 
ticular conformation, determined by the linear sequence of monomers 
in the chain. Most protein molecules and many of the RNA molecules 
found in cells fold tightly into one highly preferred conformation in this 
way (Figure 2-32). These unique conformations—shaped by evolu- 
tion—determine the chemistiy and activity of these macromolecules and 
díctate their interactions with other biological molecules. 

The noncovalent bonds important for the structure and function of mac¬ 
romolecules inelude two types described earlier: electrostatic attractions 
and hydrogen bonds (see Panel 2-7, pp. 78-79). Electrostatic attractions, 
although strong on their own, are quite weak in water because the 
charged or partially charged (polar) groups involved in the attraction are 
shielded by their interactions with water molecules and various inorganic 
ions present in the aqueous solution. Electrostatic attractions, however, 
are veiy important in biological Systems. An enzyme that binds a posi- 
tively charged substrate will often use a negatively charged amino acid 
side chain to guide its substrate into the proper position. 

Earlier, we described the importance of hydrogen bonds in determining 
the unique properties of water. They are also veiy important in the fold- 
ing of a polypeptide chain and in holding together the two strands of a 
double-stranded DNA molecule. 


In principie, there are many 
different, chemically diverse ways in 
which small molecules can be linked 
to form polymers. For example, the 
small molecule ethene (CH2=CH2) 
is used commercially to make the 
plástic polyethylene (...-CH2-CH2- 
CH2-CH2-CH2-...). The individual 
subunits of the three major classes 
of biological macromolecules, 
however, are all linked by similar 
reaction mechanisms, i.e., by 
condensation reactions that 
elimínate water. Can you think of 
any benefits that this chemistry 
offers and why it might have been 
selected in evolution? 

Figure 2-32 Most proteins and 
many RNA molecules fold into a 
particularly stable three-dimensional 
shape, or conformation. This shape is 
directed mostly by a multitude of weak, 
noncovalent intramolecular bonds. If the 
folded macromolecules are subjected to 
conditions that disrupt noncovalent bonds, 
the molecule becomes a flexible chain 
that loses both its conformation and its 
biological activity. 




3> IS /O 

a stable folded unstructured 

conformation polymer chains 

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Macromolecules in Cells 


A third type of noncovalent interaction results from van der Waals 
attractions, which are a form of electrical attraction caused by fluctuating 
electric charges that arise whenever two atoms come within a veiy short 
distance of each other. Although van der Waals attractions are weaker 
than hydrogen bonds, in large numbers they play an important role in the 
attraction between macromolecules with complementary shapes. All of 
these noncovalent bonds are reviewed in Panel 2-7, pp. 78-79. 

Another important noncovalent interaction is created by the three-dimen- 
sional structure of water, which forces together the hydrophobic portions 
of dissolved molecules in order to minimize their disruptive effect on the 
hydrogen-bonded network of water molecules (see Panel 2-7 and Panel 
2-2, pp. 68-69). This expulsión from the aqueous solution generates what 
is sometimes thought of as a fourth kind of noncovalent bond, called a 
hydrophobic interaction. Such interactions hold together phospholipid 
molecules in cell membranes, for example, and they also play a crucial 
part in the folding of protein molecules into a compact globular shape. 

Noncovalent Bonds Allow a Macromolecule to Bind Other 
Selected Molecules 

As we discussed earlier, although noncovalent bonds are individually 
weak, they can add up to create a strong attraction between two mol¬ 
ecules when these molecules fit together very closely, like a hand in a 
glove, so that many noncovalent bonds can occur between them (see 
Panel 2-7). This form of molecular interaction provides for great specifi- 
city in the binding of a macromolecule to other small and large molecules, 
because the multipoint contacts required for strong binding make it pos- 
sible for a macromolecule to select just one of the many thousands of 
different molecules present inside a cell. Moreover, because the strength 
of the binding depends on the number of noncovalent bonds that are 
formed, associations of almost any strength are possible. 

Binding of this type makes it possible for proteins to function as enzymes. 
It can also stabilize associations between any macromolecules, as long 
as their surfaces match closely (Figure 2-33 and Movie 2.4). Noncovalent 
bonds thereby allow macromolecules to be used as building blocks for 
the formation of much larger structures. For example, proteins often bind 


Why could covalent bonds not be 
used in place of noncovalent bonds 
to medíate most of the interactions 
of macromolecules? 

the surfaces of A and B, and A 
and C, are a poor match and 
are capable of forming only a few 
weak bonds; thermal motion rapidly 
breaks them apart 

the surfaces of A and D match 
well and therefore can form 
enough weak bonds to withstand 
thermal jolting; they therefore 
stay bound to each other 

Figure 2-33 Noncovalent bonds mediate interactions between macromolecules. They can also medíate interactions between 
macromolecule and small molecules (not shown). 

LibertadDigital \ 2015 


CHAPTER 2 Chemical Components of Cells 


covalent bonds noncovalent ASSEMBLY 

30 nm 

e.g., ribosome 

Figure 2-34 Both covalent bonds and noncovalent bonds are needed to form a macromolecular assembly 
such as a ribosome. Covalent bonds allow small organic molecules to join together to form macromolecules, which 
can assemble ¡nto large macromolecular complexes via noncovalent bonds. Ribosomes are large macromolecular 
machines that synthesize proteins inside cells. Each ribosome is composed of about 90 macromolecules (proteins 
and RNA molecules), and it is large enough to see ¡n the electrón microscope (see Figure 7-31). The subunits, 
macromolecules, and the ribosome here are shown roughly to scale. 

together into multiprotein complexes that function as intricate machines 
with múltiple moving parís, cariying out such complex tasks as DNA rep- 
lication and protein synthesis (Figure 2-34). In fact, noncovalent bonds 
account for a great deal of the complex chemistiy that makes life possible. 


• Living cells obey the same Chemical and physical laws as nonliving 
things. Like all other forms of matter, they are made of atoms, which 
are the smallest unit of a Chemical element that retain the distinctive 
Chemical properties of that element. 

• Cells are made up of a limited number of elements, four of which—C, 
H, N, O—make up about 96% of a cell's mass. 

• Each atom has a positively charged nucleus, which is surrounded by 
a cloud of negatively charged electrons. The Chemical properties of 
an atom are determined by the number and arrangement of its elec¬ 
trons: it is most stable when its outer electrón shell is completely 

• A covalent bond forms when a pair of outer-shell electrons is shared 
between two adjacent atoms; if two pairs of electrons are shared, a 
double bond is formed. Clusters of two or more atoms held together 
by covalent bonds are known as molecules. 

• When an electrón jumps from one atom to another, two ions of oppo- 
site charge are generated; these ions are held together by mutual 
attraction forming a noncovalent ionic bond. 

• Living organisms contain a distinctive and restricted set of small 
carbon-based (organic) molecules, which are essentially the same 
for eveiy living species. The main categories are sugars, fatty acids, 
amino acids, and nucleotides. 

• Sugars are a primaiy source of Chemical energy for cells and 
can also be joined together to form polysaccharides or shorter 

• Fatty acids are an even richer energy source than sugars, but their 
most essential function is to form lipid molecules that assemble into 
cell membranes. 

• The vast majority of the dry mass of a cell consists of macromol¬ 
ecules—mainly polysaccharides, proteins, and nucleic acids (DNA 

LibertadDigital \ 2015 

Essential Concepts 


and RNA); these macromolecules are formed as polymers of sugars, 
amino acids, or nucleotides, respectively. 

The most diverse and versatile class of macromolecules are proteins, 
which are formed from 20 types of amino acids that are covalently 
linked by peptide bonds into long polypeptide chains. 

Nucleotides play a central part in energy-transfer reactions within 
cells; they are also joined together to form information-containing 
RNA and DNA molecules, each of which is composed of only four 
types of nucleotides. 

Protein, RNA, and DNA molecules are synthesized from subunits by 
repetitive condensation reactions, and it is the specific sequence of 
subunits that determines their unique functions. 

Four types of weak noncovalent bonds—hydrogen bonds, elec- 
trostatic attractions, van der Waals attractions, and hydrophobic 
interactions—enable macromolecules to bind specifically to other 
macromolecules or to selected small molecules. 

The same four types of noncovalent bonds between different regions 
of a polypeptide or RNA chain allow these chains to fold into unique 
shapes (conformations). 



inorganic molecule 

amino acid 



ionic bond 

atomic weight 



lipid bilayer 

Avogadro's number 





molecular weight 

Chemical bond 


Chemical group 

noncovalent bond 

condensation reaction 



organic molecule 

covalent bond 

pH scale 





electrostatic attraction 


fatty a cid 


hydrogen bond 




hydronium ion 




hydrophobic interactions 

van der Waals attractions 

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Because they are polarized, two 
adjacent H 2 0 molecules can form 
a noncovalent linkage known as a 
hydrogen bond. Hydrogen bonds 
have only about 1/20 the strength 
of a covalent bond. 

Hydrogen bonds are strongest when 
the three atoms lie ¡n a straight line. 

bond lengths 

0.17 nm 

Two atoms connected by a covalent bond may exert different attractions for 
the electrons of the bond. In such cases, the bond is polar, with one end 
slightly negatively charged (5 - ) and the other slightly positively charged (8 + ). 

Although a water molecule has an overall neutral charge (havlng the same 
number of electrons and protons), the electrons are asymmetrically distributed, 
making the molecule polar. The oxygen nudeus draws electrons away from 
the hydrogen nudei, leaving the hydrogen nudei with a small net positive charge. 
The excess of electrón density on the oxygen atom creates weakly negative 
regions at the other two corners of an imaginary tetrahedron. On these pages, 
we review the Chemical properties of water and see how water ¡nfluences the 
behavior of biologlcal molecules. 


Substances that dlssolve readily in water are termed hydrophilic. They indude 
ions and polar molecules that attract water molecules through electrical charge 
effects. Water molecules surround each ion or polar molecule and carry it 
into solution. 



lonic substances such as sodium chloride 
dissolve because water molecules are 
attracted to the positive (Na + ) or negative 
(Cl“) charge of each ion. 

Polar substances such as urea 
dissolve because their molecules 
form hydrogen bonds with the 
surrounding water molecules. 


Molecules of water join together transiently 
in a hydrogen-bonded lattice. 


Jtf v 

v \ 

The cohesive nature of water is 
responsible for many of its unusual 
properties, such as high surface tensión, high 
specific heat, and high heat of vaporizaron. 


Substances that contain a preponderance 
of nonpolar bonds are usually insoluble 

in water and a 

re termed hydrophobic. 

Water molecules are not attracted to such 
hydrophobic molecules and so have little 
tendency to surround them and bring them 

into solution. 




H H 


\\ H 

\ 1 


O—. . 

\ / H 



\ / H 

o H 


H /\ 


| H H 


X H 

cX H 

Hydrocarbons, which contain many 

C-H bonds, art 

: especially hydrophobic. 

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Many substances, such as household sugar (sucrose), dissolve in water. That is, their 
molecules sepárate from each other, each becoming surrounded by water molecules. 

a n a m a °a 

a a o 6, Q #a • ° 

O O O ¡J ^ D o 
o 01 o Q a °a# & 
«• Q «a° a y 

When a substance dissolves in a 
liquid, the mixture is termed a solution. 
The dissolved substance (in this case 
sugar) is the solute, and the liquid that 
does the dissolving (in this case water) 
is the solvent. Water is an excellent 
solvent for hydrophilic substances 
because of its polar bonds. 



Substances that release hydrogen ions (protons) into solution 

Positively charged hydrogen ions (H + ) can 


are called acids. 

move from one water molecule to another, thereby creating 

two ionic species. 

HCI -► H + + 


H H H 

_ H 

hydrochloric acld hydrogen Ion 

(strong acid) 

chlorlde ion 

V / v \ © 

OS» 1 O -—± o— H 

/ ' / 

H H 

hydronium ion 


+ O 

Many of the acids important in the cell are not 

: completely 

hydroxyl ion 

dissociated, and they are therefore weak acids —for example. 

the carboxyl group (-COOFI), which dissociates 
hydrogen ion in solution. 


often written as: H 2 0 : N H + 

+ OH“ 





%ff H+ 


N oh 

(weak acid) 

O - 

Because the process is rapidly reversible, hydrogen ions are 
continually shuttling between water molecules. Puré water 
contains equal concentrations of hydronium ions and 

Note that this is a reversible reaction. 

hydroxyl ions (both 10 M). 


by the concentration 
of hydronium ions (H 3 0 + ) 
it possesses, generally 
abbreviated as H + . 

For convenience, we 
use the pH scale, where 

pH =-log 10 [H+] 

For puré water 

[H + ] = 10 7 moles/liter — 

pH = 7.0 

Substances that reduce the number of hydrogen ions in 
solution are called bases. Some bases, such as ammonia, 
combine directly with hydrogen ions. 

ammonia hydrogen ion 

ammomum io 

Other bases, such as sodium hydroxide, reduce the number of 
H + ions indirectly, by making OFT ions that then combine 
directly with H + ions to make Fl 2 0. 

NaOH — 
sodium hydroxide 

Na + 

OH - 

sodium hydroxyl 

(strong base) ion ion 

Many bases found in cells are partially associated with Fl + ions 
and are termed weak bases. This is true of compounds that 
contain an amino group (-NH 2 ), which has a weak tendency 
to reversibly accept an H + ion from water, thereby 
increasing the concentration of free OH - ions. 

-NH 2 + H + N -NH 3 + 




Monosaccharides usually have the general formula (CH 2 0) n , where n ca 
They either contain an aldehyde group (-c^ H ) and are called aldoses, c 

be 3, 4, 5, or 6, and have two o 
■ a ketone group 0e=a ) and ar 

more hydroxyl groups. 
i called ketoses. 

3-carbon (TRIOSES) 

H —C—OH 

H — C— OH 


H —C—OH 




5-carbon (PENTOSES) 

I I 

6-carbon (HEXOSES) 

H —C—OH 


HO—C —H 

H —C—OH 

& -Ife 

H —C—OH 



HO—C —H 

H —C—OH 


In aqueous solution, the aldehyde or ketone group of a 
molecule tends to react with a hydroxyl group of the sai 
molecule, thereby dosing the molecule into a ring. 



i c 





s ch 2 oh 

H D 

i c 



5 ch 2 oh 

Note that each carbón atom has a number. 

Many monosaccharides differ only in the spatial arrangement 
of atoms—that is, they are isomers. For example, glucose, 
galactose, and mannose have the same formula (C 6 H 12 0 6 ) but 
differ in the arrangement of groups around one or two carbón 

These small differences make only minor changes in the 
Chemical properties of the sugars. But the differences are 
recognized by enzymes and other proteins and therefore a 
have major biological effects. 



The hydroxyl group on the carbón that carries the 
aldehyde or ketone can rapidly change from one 
position to the other. These two positions are called 
a and p. 

e sugar is linked to another, the a o 


The hydroxyl groups of 

The carbón that carries the aldehyde 
or the ketone can react with any 
hydroxyl group on a second sugar 
molecule to form a disaccharide. 
Three common disaccharides are 
maltose (glucose + glucose) 
lactose (galactose + glucose) 
sucrose (glucose + fructose) 


Large linear and branched molecules can be made from simple repeating sugar units. 
Short chains are called oligosaccharides, and long chains are called polysaccharides. 
Glycogen, for example, is a polysaccharide made entirely of glucose units joined together. 


In many cases, a sugar sequence 
is nonrepetitive. Many different 
molecules are possible. Such 
complex oligosaccharides are 
usually linked to proteins or to lipids, 
as is this oligosaccharide, which is 
part of a cell-surface molecule 
that defines a particular blood group. 


All fatty acids have carboxyl 
groups at one end and long 
hydrocarbon tails at the other. 


ch 2 P a ' mitic ch 2 


Hundreds of different kinds of fatty acids exist. Some have one 01 
hydrocarbon tail and are said to be unsaturated. Fatty acids with 

This double bond 
is rigid and creates 
/a kink in the chain. 
The rest of the chain 
is free to rotate 
about the other C-C 


LibertadDigital \ 2015 

74 PANEL 2-5 



The common amino acids 
are grouped according to 
whether their side chains 

uncharged polar 

These 20 amino acids 

are given both three-letter 

and one-letter abbreviations. 

Thus: alanine = Ala = A 





(Lys, or K) 

(Arg, or R) 

(His, or H) 

H O 

H O 

H O 

—N —C—C — 

—N —C—C — 

i i 

—N —C—C — 

H CH, 

H CH, 

H CH 2 

ch 2 

Ch 2 This group is 
very basic 

CH 2 because its 

1 + positive charge 

nh 3 is stabilized by \ 
resonance (see 
Panel 2-1). 

ch 2 


ch 2 



í A 

[ + h 2 n nh 2 


/H^®NH + 

/ \ 

These nitrogens have a 
relatively weak affinity for an 

H + and are only partly positive 
at neutral pH. 

The general formula of an amino acid is 

, a-carbon al 

R is commonly one of 20 different side chains. 
At pH 7, both the amino and carboxyl groups 
are ionized. 

. , lr _., rnr The a-carbon atom is asymmetric, 
OPTICAL ISOMERS a ,| owing fortwo mirror y ¡ m age 

(or stereo-) isomers, L and D. 

1 *T 

I coo- I coo- 



Proteins contain exdusively L-amino acids. 


In proteins, amino acids are commonly joined together by ai 
amide linkage, called a peptide bond. 

The four atoms in each peptide bond (red box) form a rigid 
planar unit. There is no rotation around the C-N bond. 

I II I // 
N —c4c— n-Hc— c 
I I I x 

Proteins are long polymers 
of amino acids linked by 
peptide bonds, and they 
are always written with the 
N-terminus toward the left. 
Peptides are shorter, usually 
fewer than 50 amino acids long. 
The sequence of this tripeptide 
is histidine-cysteine-valine. 

+ H 3 N—C-C—N-r-C 

These two single bonds allow rotation, si 
chains of amino acids are very flexible. 

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asparticacid glutamicacid 

(Ala, or A) 

(Val, or V) 

(Asp, or D) (Glu, or E) 

H O 

H O 

_ _ 

_ ^ _|t_ 

—N —C—C — — N—C—C — 

1 1 

H CH 3 

1 1 


H CH, H CH, 

ch 3 \h 3 

c ch 2 

o cr ¿ 



o' X cr 

(Leu, or L) 

(Me, or 1) 

H O 

H O 


— N — C—C — 

h ch 2 

I I; 




/ \ 

ch 3 ch 2 


/ \ 

ch 3 ch 3 


ch 3 



asparagine glutamine 

(Pro, or P) 

(Phe, or F) 

(Asn, orN) (Gln, or Q) 

H O 

H O 

H O H O 

| 1 

y I I 


— N — C—C — 

— N —C—C— —N —C —C — 


/ \ 

CH, CH, 

1 1 

H CH 2 

H CH 2 H CH 2 


| | 

(actuallyan CH 2 


C CH, 

imino acid) 

S \ | 

O NH, X 

\ c¡ nh 2 



\ / 

(Met, or M) 

(Trp, or W) 


H O 

1 II 

H O 

1 II 

Although the amide N is not charged at 


| | 

—N—C—C — 

neutral pH, it is polar. 

1 1 

H CH 2 

H CH 2 


[i -J 1 

S— ch 3 

serine threonine tyrosine 


(Ser, or S) (Thr, or T) (Tyr, or Y) 

H O H O H O 

i ii i ii ¿Ai ^ 



(Gly, or G) 

(Cys, or C) 

—N—C—C— —N—C—C— —N—C—C— 

II II i 1, 

H CH 2 H CH—CH 3 H CH 2 

H O 

1 II 

H O 

1 f 

1 1 ' I 

—N —C—C — 

—N—C—C — 


X / T 

1 1 

H H 

1 1 

H CH 2 

\ /X . OH 


Disulfide bonds can form between two cysteine side chains 

The-OH group is polar. 

in proteins. 

- CH 2 — s— s 

1 —ch 2 - - 



The phosphates are normally joined to 
the C5 hydroxyl of the ribose or 
deoxyribose sugar (designated 5')- Mono-, 
di-, and triphosphates are common. 


“O— P- 

~ 0 ~P AMP 





“O— P- 

_o-P-o-CH 2 “ ¡ J 



M f 

~o — P— 

_o-p-o-p-o-ch 2 


Or Q- 

The phosphate makes a nudeotide 
negatively charged. 

A nudeotide consists of a nitrogen-containing 
base, a five-carbon sugar, and one or more 
phosphate groups. 



The base is linked to 
the same carbón (C1) 
used in sugar-sugar 

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NOMENCLATURE The ñames can be confusing, butthe abbreviations are dear. 



















Nudeotides are abbreviated by 
three capital letters. Some examples 

AMP = adenosine monophosphate 
dAMP = deoxyadenosine monophosphate 
UDP = uridine diphosphate 
ATP = adenosine triphosphate 



Nudeotides are joined together by 
phosphodiester bonds between 5' and 
3' carbón atoms of the sugar ring, via a 
I phosphate group, to form nudeic acids. 
I The linear sequence of nudeotides in a 
1 nudeic acid Chain is commonly 
abbreviated by a one-letter code, such 
¡s AGCTTACA, with the 5' end of the 

3' end of chain 


They carry Chemical energy in their easily hydrolyzed phosphoanhydride bonds. 

NH 2 

phosphoanhydride bonds 


“O— P -Í-O— P —O — P — O — CH, 


example: ATP (or ) 

2 | They combine with other groups to form coenzymes. 



HS—C -C —N -C —C -C —N —C —C —C — C P —O— P J-O — CH, 

iii iii mi nw" 

H H H H H H HO CH, H 0~ ( 

example: coenzyme A (CoA) 


3 ( They are used as small intracellular signaling molecules in the cell. 
example: cydic AMP 





Organic molecules can ¡nteract with other molecules through 
three types of short-range attractive forces known as 
noncovalent bonds'. van der Waals attractions, electrostatic 
attractions, and hydrogen bonds. The repulsión of 
hydrophobic groups from water is also important for these 
interactions and for the folding of biological macromolecules. 

Weak noncovalent bonds have less than 1/20 the strength of 
a strong covalent bond. They are strong enough to provide 
tight binding only when many of them are formed 


As already described for water (see Panel 2-2, pp. 68-69), 
hydrogen bonds form when a hydrogen atom ¡s 
"sandwiched" between two electron-attracting atoms 
(usually oxygen or nitrogen). 

Hydrogen bonds are strongest when the three atoms are 
in a straight line: 


If two atoms are too cióse together they repel each other 
very strongly. For this reason, an atom can often be 
treated as a sphere with a fixed radius. The characteristic 
"size" for each atom is specified by a unique van der 
Waals radius. The contact distance between any two 
noncovalently bonded atoms is the sum of their van der 
Waals radii. 

^ t i 

0.12 nm 0.2 nm 0.15 nm 0.14 nm 

radius radius radius radius 

At very short distances, any two atoms show a weak 
bonding interaction due to their fluctuating electrical 
charges. The two atoms will be attracted to each other 
in this way until the distance between their nudei is 
approximately equal to the sum of their van der Waals 
radii. Although they are ¡ndividually very weak, such 
van der Waals attractions can become important when 
two macromolecular surfaces fit very cióse together, 
because many atoms are involved. 

Note that when two atoms form a covalent bond, the 
centers of the two atoms (the two atomic nudei) are 
much doser together than the sum of the two van der 
Waals radii. Thus, 

0.4 nm 0.15 nm 0.13 nm 

two non-bonded two carbón two carbón 

carbón atoms atoms held by atoms held by 

Examples in macromolecules: 

Amino acids in a polypeptide chain can be hydrogen-bonded 
together in a folded protein. 


Any two atoms that can form hydrogen bonds to each other 
can alternatively form hydrogen bonds to water molecules. 
Because of this competition with water molecules, the 
hydrogen bonds formed in water between two peptide bonds, 
for example, are relatively weak. 


R—C —H 

' 1 

c o I 


H—C —R 


Two bases, G and C, are hydrogen-bonded 



CMC Gs-iC 

// \ / \ 


2H 2 0 i 


2H 2 0 

LibertadDigital \ 2015 



Attractive interactions occur both between 
fully charged groups (¡onic bond) and between 
partially charged groups on polar molecules. 


Charged groups are shielded by their 
interactions with water molecules. 
Electrostatic attractions are therefore 
quite weak in water. 

iiii/io y 

Km ,/ H %0 ^ h H 

The forcé of attraction between the two partial 
charges, 5 + and 8 _ , falls off rapidly as the 
distance between the charges ¡ncreases. 

In the absence of water, ionic bonds are very strong. 
They are responsible for the strength of such 
minerals as marble and agate, and for crystal 
formation in common table salt, NaCI. 

Inorganic ions in solution can also duster around 
charged groups and further weaken these electrostatic 

a crystal of 


- - 1 . . ( 

,** * " i 

•ÍW * 

m ci^ 

Despite being weakened by water and inorganic 
ions, electrostatic attractions are very important 
in biological Systems. For example, an enzyme 
that binds a positively charged substrate will 
often have a negatively charged amino acid side 
Chain at the appropriate place. 

Water forces hydrophobic groups together 

in order to minimize their disruptive 

effects on the water network formed by the H bonds 

between water molecules. Hydrophobic groups 

held together in this way are sometimes said 

to be held together by "hydrophobic 

bonds," even though the attraction is 

actually caused by a repulsión from water. 


CHAPTER 2 Chemical Components of Cells 



Which of the following statements are corred? Explain your 

A. An atomic nucleus contains protons and neutrons. 

B. An atom has more eledrons than protons. 

C. The nucleus is surrounded by a double membrane. 

D. All atoms of the same element have the same number 
of neutrons. 

E. The number of neutrons determines whether the 
nucleus of an atom is stable or radioadive. 

F. Both fatty acids and polysaccharides can be important 
energy stores in the cell. 

G. Hydrogen bonds are weak and can be broken by 
thermal energy, yet they contribute significantly to the 
specificity of interadions between macromolecules. 


To gain a better feeling for atomic dimensions, assume that 
the page on which this question is printed is made entirely 
of the polysaccharide cellulose, whose molecules are 
described by the formula (C n H2 n O n ), where n can be a quite 
large number and is variable from one molecule to another. 
The atomic weights of carbón, hydrogen, and oxygen are 
12, 1, and 16, respedively, and this page weighs 5 g. 

A. How many carbón atoms are there in this page? 

B. In cellulose, how many carbón atoms would be stacked 
on top of each other to span the thickness of this page (the 
size of the page is 21.2 cm X 27.6 cm, and it is 0.07 mm 

C. Now consider the problem from a different angle. 
Assume that the page is composed only of carbón atoms. 

A carbón atom has a diameter of 2 X 10~ 10 m (0.2 nm); how 
many carbón atoms of 0.2 nm diameter would it take to 
span the thickness of the page? 

D. Compare your answers from parts B and C and explain 
any differences. 


A. How many eledrons can be accommodated in the first, 
second, and third eledron shells of an atom? 

B. How many eledrons would atoms of the elements listed 
below have to gain or lose to obtain a completely filled 
outer shell? 

helium gain_ lose_ 

oxygen gain_ lose_ 

carbón gain_ lose_ 

sodium gain_ lose_ 

chlorine gain_ lose_ 

C. What do the answers tell you about the readivity of 
helium and the bonds that can form between sodium and 


The elements oxygen and sulfur have similar Chemical 
properties because they both have six eledrons in their 
outermost electrón shells. Indeed, both elements form 
molecules with two hydrogen atoms, water (H2O) and 
hydrogen sulfide (H2S). Surprisingly, at room temperature, 
water is a liquid, yet H2S is a gas, despite sulfur being much 
larger and heavier than oxygen. Explain why this might be 
the case. 


Write the Chemical formula for a condensation readion of 
two amino acids to form a peptide bond. Write the formula 
for its hydrolysis. 


Which of the following statements are corred? Explain your 

A. Proteins are so remarkably diverse because each is 
made from a unique mixture of amino acids that are linked 
in random order. 

B. Lipid bilayers are macromolecules that are made up 
mostly of phospholipid subunits. 

C. Nucleic acids contain sugar groups. 

D. Many amino acids have hydrophobic side chains. 

E. The hydrophobic tails of phospholipid molecules are 
repelled from water. 

F. DNA contains the four different bases A, G, U, and C. 

A. How many different molecules composed of (a) two, 

(b) three, and (c) four amino acids, linked together by 
peptide bonds, can be made from the set of 20 naturally 
occurring amino acids? 

B. Assume you were given a mixture consisting of one 
molecule each of all possible sequences of a smallish 
protein of molecular weight 4800 daltons. If the average 
molecular weight of an amino acid is, say, 120 daltons, how 
much would the sample weigh? How big a container would 
you need to hold it? 

C. What does this calculation tell you about the fraction 
of possible proteins that are currently in use by living 
organisms (the average molecular weight of proteins is 
about 30,000 daltons)? 


This is a biology textbook. Explain why the Chemical 
principies that are described in this chapter are important 
in the context of modern cell biology. 


A. Describe the similarities and differences between van 
der Waals attractions and hydrogen bonds. 

LibertadDigital \ 2015 

B. Which of the two bonds would form (a) between two 
hydrogens bound to carbón atoms, (b) between a nitrogen 
atom and a hydrogen bound to a carbón atom, and 
(c) between a nitrogen atom and a hydrogen bound to 
an oxygen atom? 


What are the forces that determine the folding of a 
macromolecule into a unique shape? 

Chapter 2 End-of-Chapter Questions 81 


Fatty acids are said to be "amphipathic." What is meant by 
this term, and how does an amphipathic molecule behave 
in water? Draw a diagram to ¡Ilústrate your answer. 


Are the formulas in Figure Q2-21 corred or incorred? 
Explain your answer in each case. 



H,N —c—COOH 



o© o© o© 

CH 3 —CH 2 —OH 


8 + 5“ 5 + 


Figure Q2-21 





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left intentionally blank 

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* 4 « 


Energy, Catalysis, and 

One property above all makes living things seem almost miraculously 
different from nonliving matter: they create and maintain order in a uni- 
verse that is tending always toward greater disorder. To accomplish this 
remarkable feat, the cells in a living organism must carry out a never- 
ending stream of Chemical reactions that produce the molecules the 
organism requires to meet its metabolic needs. In some of these reac¬ 
tions, small organic molecules—amino acids, sugars, nucleotides, and 
lipids—are taken apart or modified to supply the many other small mol¬ 
ecules that the cell requires. In other reactions, these small molecules 
are used to construct an enormously diverse range of larger molecules, 
including the proteins, nucleic acids, and other macromolecules that 
endow living systems with all of their most distinctive properties. Each 
cell can be viewed as a tiny Chemical factoiy, performing many millions 
of these reactions every second. 

To carry out the tremendous number of Chemical reactions needed to 
sustain it, a living organism requires both a source of atoms in the form 
of food molecules and a source of energy. The atoms and the energy must 
both come, ultimately, from the nonliving environment. In this chapter, 
we discuss why cells require energy, and how they use energy and atoms 
from their environment to create the molecular order that makes life 

Most of the Chemical reactions that cells perform would normally occur 
only at temperatures that are much higher than those inside a cell. Each 
reaction therefore requires a major boost in Chemical reactivity to enable 
it to proceed rapidly within the cell. This boost is provided by specialized 
proteins called enzymes, each of which accelerates, or catalyzes, just one 




LibertadDigital \ 2015 


CHAPTER 3 Energy, Catalysis, and 

Figure 3-1 A series of enzyme- 
catalyzed reactions forms a metabolic 
pathway. Each enzyme catalyzes a 
Chemical reaction involving a particular 
molecule. In this example, a set of 
enzymes acting in series converts 
molecule A to molecule F, forming a 
metabolic pathway. 

food the many molecules 

molecules that form the cell 

the many building blocks 
for biosynthesis 

Figure 3-2 Catabolic and anabolic 
pathways together constitute the cell's 
metabolism. Note that a major portion of 
the energy stored in the Chemical bonds of 
food molecules ¡s dissipated as heat. Thus, 
only some of this energy can be converted 
to the useful forms of energy needed to 
drive the synthesis of new molecules. 


molecule molecule molecule molecule molecule molecule 


catalysis by 
enzyme 1 


catalysis by 
enzyme 2 

catalysis by 
enzyme 3 


catalysis by 
enzyme 4 


catalysis by 
enzyme 5 

of the many possible kinds of reactions that a particular molecule might 
undergo. These enzyme-catalyzed reactions are usually connected in 
series, so that the product of one reaction becomes the starting material 
for the next (Figure 3-1). The long linear reaction pathways, or metabolic 
pathways, that result are in turn linked to one another, forming a complex 
web of interconnected reactions. 

Rather than being an inconvenience, the necessity for catalysis is a ben- 
efit, as it allows the cell to precisely control its metabolism— the sum 
total of all the Chemical reactions it needs to carry out to survive, grow, 
and reproduce. This control is central to the chemistry of life. 

Two opposing streams of Chemical reactions occur in cells, the catabolic 
pathways and the anabolic pathways. The catabolic pathways (catabo- 
lism) break down foodstuffs into smaller molecules, thereby generating 
both a useful form of energy for the cell and some of the small molecules 
that the cell needs as building blocks. The anabolic, or biosynthetic, path¬ 
ways (anabolism) use the energy harnessed by catabolism to drive the 
synthesis of the many molecules that form the cell. Together, these two 
sets of reactions constitute the metabolism of the cell (Figure 3-2). 

The details regarding the individual reactions that comprise cell metabo¬ 
lism are part of the subject matter of biochemistiy, and they need not 
concern us here. But the general principies by which cells obtain energy 
from their environment and use it to create order are central to cell biol- 
ogy. We begin this chapter with a discussion of why a constant input 
of energy is needed to sustain living organisms. We then discuss how 
enzymes catalyze the reactions that produce biological order. Finally, we 
describe the molecules that carry the energy that makes life possible. 


Nonliving things left to themselves eventually become disordered: build- 
ings crumble and dead organisms decay. Living cells, by contrast, not 
only maintain, but actually generate order at every level, from the large- 
scale structure of a butterfly or a flower down to the organization of the 
molecules that make up these organisms (Figure 3-3). This property of 
life is made possible by elabórate molecular mechanisms that extract 
energy from the environment and convert it into the energy stored in 
Chemical bonds. Biological structures are therefore able to maintain their 
form, even though the materials of which they are made are continu- 
ally being broken down, replaced, and recycled. Your body has the same 
basic structure it had 10 years ago, even though you now contain atoms 
that, for the most part, were not in your body then. 

Biological Order Is Made Possible by the Release of 
Heat Energy from Cells 

The universal tendency of things to become disordered is expressed in a 
fundamental law of physics, the second law of thermodynamics. This law 
States that, in the universe or in any isolated System (a collection of mat¬ 
ter that is completely isolated from the rest of the universe), the degree 
of disorder can only increase. The second law of thermodynamics has 
such profound implications for living things that it is worth restating in 
several ways. 

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The Use of Energy by Cells 


We can express the second law in terms of probability by stating that 
Systems will change spontaneously toward those arrangements that have 
the greatestprobability. Consider a box of 100 coins all lying heads up. A 
series of events that disturbs the box will tend to move the arrangement 
toward a mixture of 50 heads and 50 tails. The reason is simple: there are 
a huge number of possible arrangements of the individual coins that can 
achieve the 50-50 result, but only one possible arrangement that keeps 
them all oriented heads up. Because the 50-50 mixture accommodates a 
greater number of possibilities and places fewer constraints on the ori- 
entation of each individual coin, we say that it is more "disordered." For 
the same reason, one's living space will become increasingly disordered 
without an intentional effort to keep it organized. Movement toward dis- 
order is a spontaneous process, requiring a periodic input of energy to 
reverse it (Figure 3-4). 

The measure of a system's disorder is called the entropy of the System, 
and the greater the disorder, the greater the entropy. Thus another way 
to express the second law of thermodynamics is to say that Systems 
will change spontaneously toward arrangements with greater entropy. 
Living cells—by surviving, growing, and forming complex communities 
and even whole organisms—generate order and thus might appear to 
defy the second law of thermodynamics. This is not the case, however, 

Figure 3-3 Biological structures are 
highly ordered. Well-defined, órnate, and 
beautiful spatial patterns can be found 
at every level of organizaron in living 
organisms. In order of increasing size: 

(A) protein molecules in the coat of a virus 
(a parasite that, although nottechnically 
alive, contains the same types of molecules 
as those found in living cells); (B) the regular 
array of microtubules seen in a cross section 
of a sperm tai!; (C) surface contours of a 
pollen grain (a single cell); (D) cross section 
of a fern stem, showing the patterned 
arrangement of cells; and (E) flower with a 
spiral array of petáis, each made of milllons 
of cells. (A, courtesy of Robert Grant, 
Stéphane Crainic, and James M. Hogle; 

B, courtesy of Lewis Tilney; C, courtesy of 
Colín MacFarlane and Chris Jeffree; 

D, courtesy of Jim Haseloff.) 


Figure 3-4 The spontaneous tendency 
toward disorder is an everyday 
experience. Reversing this natural tendency 
toward disorder requires an intentional 
effort and an input of energy. In fact, from 
the second law of thermodynamics, we 
can be certain that the human intervention 
requlred will release enough heat to the 
environment to more than compénsate for 
the reestablishment of order in this room. 

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CHAPTER 3 Energy, Catalysis, and Biosynthesis 

Figure 3-5 Living cells do not defy the 
second law of thermodynamics. In the 

diagram on the left, the molecules of both 
the cell and the rest of the universe (the 
environment) are depicted in a relatlvely 
disordered State. In the diagram on the 
right, the cell has taken in energy from food 
molecules and released heat by carrylng out 
a reactlon that orders the molecules that the 
cell contalns. Because the heat ¡ncreases 
the disorder in the environment around the 
cell—as depicted by the longer, jagged red 
arrows, whlch represent ¡ncreased thermal 
motlon, and the distorted molecules, 
whlch Indícate enhanced molecular 
vibration and rotatlon—the second law of 
thermodynamics is satisfled, even as the cell 
grows and constructs larger molecules. 

because a cell is not an isolated System. Rather, it takes in energy from 
its environment—in the form of food, inorganic molecules, or photons of 
light from the sun—and it then uses this energy to generate order within 
itself, forging new Chemical bonds and building large macromolecules. 
In the course of performing the Chemical reactions that generate order, 
some energy is lost in the form of heat. Heat is energy in its most disor¬ 
dered form—the random jostling of molecules (analogous to the random 
jostling of the coins in the box). Because the cell is not an isolated Sys¬ 
tem, the heat energy that its reactions generate is quickly dispersed into 
the cell’s surroundings. There, the heat increases the intensity of the ther¬ 
mal motions of nearby molecules, thereby increasing the entropy of the 
environment (Figure 3-5). 

The amount of heat released by a cell must be great enough that the 
increased order generated inside the cell is more than compensated for 
by the increased disorder generated in the environment. Only in this case 
is the second law of thermodynamics satisfled, because the total entropy 
of the System—that of the cell plus its environment—increases as a result 
of the Chemical reactions inside the cell. 

Cells Can Convert Energy from One Form to Another 

According to the flrstlaw of thermodynamics, energy cannot be created or 
destroyed—but it can be converted from one form to another (Figure 3-6). 
Cells take advantage of this law of thermodynamics, for example, when 
they convert the energy from sunlight into the energy in the Chemical 
bonds of sugars and other small organic molecules during photosynthe- 
sis. Although Chemical reactions that power such energy conversions can 
change how much energy is present in one form or another, the first law 
tells us that the total amount of energy in the universe must always be 
the same. 

When an animal cell breaks down foodstuffs, some of the energy in 
the Chemical bonds in the food molecules (chemical-bond energy) is 
converted into the thermal motion of molecules (heat energy). This con¬ 
versión of Chemical energy into heat energy causes the universe as a 
whole to become more disordered—as required by the second law of ther¬ 
modynamics. But the cell cannot derive any benefit from the heat energy 
it produces unless the heat-generating reactions are directly linked to 
processes that maintain molecular order inside the cell. It is the tight 
coupling of heat production to an increase in order that distinguishes the 
metabolism of a cell from the wasteful burning of fuel in a fire. Later in 
this chapter, we ¡Ilústrate how this coupling occurs. For the moment, it is 

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The Use of Energy by Cells 


has potential 
energy due 
to pulí of 

falling brlck has 
kinetic energy 

fP % 

heat ¡s released 
the floor 

A potential energy due to position-► kinetic energy 

4 * 


rapid vibrations and 
rotations of two newly 
formed water molecules 

B chemical-bond energy in H 2 and 0 2 

rapid molecular 
motions in H 2 0 
(kinetic energy) 

heat dispersed to 

C chemical-bond energy -► electrical energy -► kinetic energy 

sunlight chlorophyll chlorophyll molecule 

molecule in excited State photosynthesis 

D electromagnetic (light) energy -► high-energy electrons -► chemical-bond energy 

sufficient to recognize that—by directly linking the "burning" of food mol¬ 
ecules to the generation of biological order—cells are able to create and 
maintain an island of order in a universe tending toward chaos. 

Photosynthetic Organisms Use Sunlight to Synthesize 
Organic Molecules 

All animáis live on energy stored in the Chemical bonds of organic mol¬ 
ecules, which they take in as food. These food molecules also provide the 
atoms that animáis need to construct new living matter. Some animáis 
obtain their food by eating other animáis, others by eating plants. Plants, 
by contrast, obtain their energy directly from sunlight. Thus, the energy 
animáis obtain by eating plants—or by eating animáis that have eaten 
plants—ultimately comes from the sun (Figure 3-7). 

Solar energy enters the living world through photosynthesis, a process 
that converts the electromagnetic energy in sunlight into chemical-bond 
energy in cells. Photosynthetic organisms—including plants, algae, and 

Figure 3-6 Different forms of energy 
are interconvertible, but the total amount 
of energy must be conserved. In (A), we 
can use the height and weight of the bride 
to predict exactly how much heat will be 
released when ¡t hits the floor. In (B), the 
large amount of chemical-bond energy 
released when water (H2O) ¡s formed from 
H2 and O2 is initially converted to very 
rapid thermal motions in the two new H2O 
molecules; however, collisions with other 
H2O molecules almost instantaneously 
spread this kinetic energy evenly throughout 
the surroundings (heat transfer), making 
the new H2O molecules indistinguishable 
from all the rest. (C) Cells can convert 
chemical-bond energy into kinetic energy 
to drive, for example, molecular motor 
proteins; however, this occurs without the 
intermedíate conversión to electrical energy 
that a man-made appliance such as this fan 
requires. (D) Some cells can also harvest 
the energy from sunlight to form Chemical 
bonds vía photosynthesis. 

Figure 3-7 With few exceptions, the 
radiant energy of sunlight sustains 
all life. Trapped by plants and some 
microorganisms through photosynthesis, 
light from the sun is the ultímate source of 
all energy for humans and other animáis. 

(Wheat Field Behind Saint-Paul Hospital 
with a Reaper by Vincent van Gogh. 
Courtesy of Museum Folkwang, Essen.) 

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CHAPTER 3 Energy, Catalysis, and Biosynthesis 

h 2 o mt 


STAGE 1 - 1 1 - STAGE 2 

Figure 3-8 Photosynthesis takes place 
in two stages. The activated carriers 
generated in the first stage are two 
molecules that we will discuss shortly: 


Consider the equation 

light energy + CO2 + H2O —> 
sugars + O2 + heat energy 
Would you expect this reaction to 
occur in a single step? Why must 
heat be generated in the reaction? 
Explain your answers. 

some bacteria—use the energy they derive from sunlight to synthesize 
small Chemical building blocks such as sugars, amino acids, nucleotides, 
and fatty acids. These small molecules in turn are converted into the 
macromolecules—the proteins, nucleic acids, polysaccharides, and lip- 
ids—that form the plant. 

We describe the elegant mechanisms that underlie photosynthesis in 
detail in Chapter 14. Generally speaking, the reactions of photosynthe¬ 
sis take place in two stages. In the first stage, energy from sunlight is 
captured and transiently stored as chemical-bond energy in specialized 
molecules called activated carriers, which we discuss in more detail later 
in the chapter. All of the oxygen (O 2 ) in the air we breathe is generated by 
the splitting of water molecules during this first stage of photosynthesis. 
In the second stage, the activated carriers are used to help drive a carbon- 
Jkation process, in which sugars are manufactured from carbón dioxide 
gas (CO 2 ). In this way, photosynthesis generates an essential source of 
stored chemical-bond energy and other organic materials—for the plant 
itself and for any animáis that eat it. The two stages of photosynthesis are 
summarized in Figure 3-8. 

Cells Obtain Energy by the Oxidation of Organic 

All animal and plant cells require the Chemical energy stored in the 
Chemical bonds of organic molecules—either the sugars that a plant has 
produced by photosynthesis as food for itself or the mixture of large and 
small molecules that an animal has eaten. To use this energy to live, 
grow, and reproduce, organisms must extract it in a usable form. In both 
plants and animáis, energy is extracted from food molecules by a process 
of gradual oxidation, or controlled buming. 

Earth's atmosphere is about 21% oxygen. In the presence of oxygen, the 
most energetically stable form of carbón is CO 2 and that of hydrogen is 
H 2 O. A cell is therefore able to obtain energy from sugars or other organic 
molecules by allowing the carbón and hydrogen atoms in these mole¬ 
cules to combine with oxygen—that is, become oxidizcd —to produce CO 2 
and H 2 O, respectively—a process known as cellular respiration. 
Photosynthesis and cellular respiration are complementary processes 
(Figure 3-9). This means that the transactions between plants and ani¬ 
máis are not all one way. Plants, animáis, and microorganisms have 
existed together on this planet for so long that they have become an 
essential part of each other's environments. The oxygen released by 
photosynthesis is consumed by nearly all organisms for the oxidative 
breakdown of organic molecules. And some of the CO 2 molecules that 
today are incorporated into organic molecules by photosynthesis in a 
green leaf were released yesterday into the atmosphere by the respi¬ 
ration of an animal, a fungus, or the plant itself, or by the burning of 

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The Use of Energy by Cells 


' C0 2 + H 2 0 — 0 2 + SUGARS ' SUGARS + 0 2 —H 2 0 + C0 2 ' 

fossil fuels. Carbón utilization therefore forms a huge cycle that involves 
the biosphere (all of the living organisms on Earth) as a whole, Crossing 
boundaries between individual organisms (Figure 3-10). 

Oxidation and Reduction Involve Electron Transfers 

The cell does not oxidize organic molecules in one step, as occurs when 
organic material is burned in a fire. Through the use of enzyme catalysts, 
metabolism directs the molecules through a large number of reactions, 
few of which actually involve the direct addition of oxygen. Thus, before 
we consider some of these reactions, we should explain what is meant 
by oxidation. 

The term oxidation literally means the addition of oxygen atoms to a mol- 
ecule. More generally, though, oxidation is said to occur in any reaction 
in which electrons are transferred frorn one atom to another. Oxidation, 
in this sense, refers to the removal of electrons frorn an atom. The con¬ 
verse reaction, called reduction, involves the addition of electrons to an 
atom. Thus, Fe 2+ is oxidized when it loses an electrón to become Fe 3+ , 
whereas a chlorine atom is reduced when it gains an electrón to become 
Ch. Because the number of electrons is conserved in a Chemical reac¬ 
tion (there is no net loss or gain), oxidation and reduction always occur 
simultaneously: that is, if one molecule gains an electrón in a reaction 
(reduction), a second molecule must lose the electrón (oxidation). When 
a sugar molecule is oxidized to CO 2 and H 2 O, for example, the O 2 mol¬ 
ecules involved in forming H 2 O gain electrons and thus are said to have 
been reduced. 

The terms oxidation and reduction apply even when there is only a partial 
shift of electrons between atoms linked by a covalent bond. When a car¬ 
bón atom becomes covalently bonded to an atom with a strong affinity 
for electrons—oxygen, chlorine, or sulfur, for example—it gives up more 

Figure 3-9 Photosynthesis and cellular 
respiration are complementary processes 
in the living world. The /eftside ofthe 
diagram shows how photosynthesis— 
carried out by plants and photosynthetic 
microorganisms—uses the energy of 
sunlight to produce sugars and other 
organic molecules frorn the carbón 
atoms in CO2 in the atmosphere. In 
turn, these molecules serve as food for 
other organisms. The right side ofthe 
diagram shows how cellular respiration 
in most organisms—including plants and 
photosynthetic microorganisms—uses O2 
to oxidize food molecules, releasing the 
same carbón atoms in the form of CO2 
backto the atmosphere. In the process, 
the organisms obtain the useful chemical- 
bond energy that they need to survive. 

The first cells on Earth are thought to have 
been capable of neither photosynthesis ñor 
cellular respiration (discussed in Chapter 
14). However, photosynthesis must have 
preceded respiration on the Earth, because 
there is strong evidence that billions of 
years of photosynthesis were required to 
release enough C>2to create an atmosphere 
that could support respiration. 

Figure 3-10 Carbón atoms cycle 
continuously through the biosphere. 

Individual carbón atoms are ¡ncorporated 
into organic molecules of the living world by 
the photosynthetic activity of plants, algae, 
and bacteria. They then pass to animáis 
and microorganisms—as well as into 
organic material in soil and oceans—and 
are ultimately restored to the atmosphere 
in the form of CO2 when organic molecules 
are oxidized by cells during respiration or 
burned by humans as fossil fuels. 

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CHAPTER 3 Energy, Catalysis, and Biosynthesis 




partial / 
positive ^ 
charge (S + ) 

x negative 
charge (S~) 

Figure 3-11 Oxidation and reduction ¡nvolve a shift in the balance of electrons. 

(A) When two atoms form a polar covalent bond (as discussed ¡n Chapter 2, p. 44), the 
atom that ends up with a greater share of electrons (represented by the blue clouds) 
is said to be reduced, while the other atom, with a lesser share of electrons, is said to 
be oxidized. The reduced atom has acquired a partial negative charge (8 _ ); conversely, 
the oxidized atom has acquired a partial positive charge (8 + ), as the positive charge 
on the atomic nucleus now exceeds the total charge of the electrons surrounding it. 

(B) A simple reduced carbón compound, such as methane, can be oxidized in a stepwise 
fashion by the successive replacement of its covalently bonded hydrogen atoms with 
oxygen atoms. With each step, electrons are shifted away from the carbón, and the 
carbón atom becomes progressively more oxidized. Moving in the opposite direction, 
carbón dioxide becomes progressively more reduced as its oxygen atoms are replaced 
by hydrogens to yield methane. 

than its equal share of electrons and forms a polar covalent bond. The 
positive charge of the carbón nucleus now slightly exceeds the negative 
charge of its electrons, so that the carbón atom acquires a partial positive 
charge (8 + ) and is said to be oxidized. Conversely, the carbón atom in a 
C-H bond has somewhat more than its share of electrons; it acquires a 
partial negative charge (8“), and so is said to be reduced (Figure 3-11 A). 
When a molecule in a cell picks up an electrón (e~), it often picks up a 
protón (H + ) at the same time (protons being freely available in water). 
The net effect in this case is to add a hydrogen atom to the molecule: 

A + e- + H + -> AH 

Even though a proton plus an electrón is involved (instead of just an 
electrón), such hydrogenation reactions are reductions, and the reverse, 
dehydrogenation, reactions are oxidations. An easy way to tell whether an 
organic molecule is being oxidized or reduced is to count its C-H bonds: 
reduction occurs when the number of C-H bonds increases, whereas oxi¬ 
dation occurs when the number of C-H bonds decreases (Figure 3-11B). 
As we will see later in this chapter—and again in Chapter 13—cells use 
enzymes to catalyze the oxidation of organic molecules in small steps, 
through a sequence of reactions that allows energy to be harvested in 
useful forms. 


Enzymes, like cells, obey the second law of thermodynamics. Although 
they can speed up energetically favorable reactions—those that produce 
disorder in the universe—enzymes cannot by themselves forcé energeti¬ 
cally unfavorable reactions to occur. Cells, however, must do just that 
in order to grow and divide—or just to survive. They must build highly 
ordered and energy-rich molecules from small and simple ones—a proc- 
ess that requires an input of energy. 

To understand how enzymes promote catalysis—the acceleration of 
the specific Chemical reactions needed to sustain life—we first need to 
examine the energetics involved. In this section, we consider how the 
free energy of molecules contributes to their chemistry, and we see how 

LibertadDigital \ 2015 

Free Energy and Catalysis 


free-energy changes—which reflect how much total disorder is generated 
in the universe by a reaction—influence whether and how the reaction 
will proceed. We then discuss how enzymes lower the activation energy 
needed to initiate reactions in the cell. And we describe how enzymes 
can exploit differences in the free-energy changes of different reactions 
to drive the energetically unfavorable reactions that produce biological 
order. Such enzyme-assisted catalysis is crucial for cells: without it, life 
could not exist. 

Chemical Reactions Proceed in the Direction that Causes 
a Loss of Free Energy 

Paper bums readily, releasing into the atmosphere water and carbón 
dioxide as gases, while simultaneously releasing energy as heat: 

paper + 0 2 —> smoke + ashes + heat + CO 2 + H 2 0 
This reaction occurs in only one direction: smoke and ashes never spon- 
taneously gather carbón dioxide and water from the heated atmosphere 
and reconstitute themselves into paper. When paper burns, much of its 
Chemical energy is dissipated as heat: it is not lost from the universe, 
since energy can never be created or destroyed; instead, it is irretrievably 
dispersed in the chao tic random thermal motions of molecules. At the 
same time, the atoms and molecules of the paper become dispersed and 
disordered. In the language of thermodynamics, there has been a release 
of free energy —that is, energy that can be harnessed to do work or drive 
Chemical reactions. This release reflects a loss of orderliness in the way 
the energy and molecules had been stored in the paper. We will discuss 
free energy in more detail shortly, but the general principie can be sum- 
marized as follows: Chemical reactions proceed only in the direction that 
leads to a loss of free energy. In other words, the spontaneous direction 
for any reaction is the direction that goes "downhill." A "downhill" reac¬ 
tion in this sense is said to be energetically favorable. 

Enzymes Reduce the Energy Needed to Initiate 
Spontaneous Reactions 

Although the most energetically favorable form of carbón under ordi- 
nary conditions is CO 2 , and that of hydrogen is H 2 O, a living organism 
will not disappear in a puff of smoke, and the book in your hands will 
not burst spontaneously into flames. This is because the molecules in 
both the living organism and the book are in a relatively stable State, and 
they cannot be changed to lower-energy States without an initial input 
of energy. In other words, a molecule requires a boost over an energy 
barrier before it can undergo a Chemical reaction that moves it to a lower- 
energy (more stable) State (Figure 3-12A). This boost is known as the 

reaction pathway reaction pathway 


In which of the following reactions 
does the red atom undergo an 

A. Na — ► Na + (Na atom —> Na + ion) 

B. CI->C|- (Cl atom —► Cl~ ion) 


(ethanol —► acetaldehyde) 

D. CH3CHO —» CH3COO - 

(acetaldehyde —> acetic acid) 

E. CH 2 =CH 2 —» CH 3 CH 3 

(ethene —> ethane) 

Figure 3-12 Even energetically favorable 
reactions require activation energy to get 
them started. (A) Compound Y (a reactant) 
is in a relatively stable State; thus energy 
is required to convert it to compound X 
(a product), even though X is at a lower 
overall energy level than Y. This conversión 
will not take place, therefore, unless 
compound Y can acquire enough activation 
energy (energy a minus energy b) from its 
surroundings to undergo the reaction that 
converts it into compound X. This energy 
may be provided by means of an unusually 
energetic collision with other molecules. For 
the reverse reaction, X —► Y, the activation 
energy required will be much larger (energy 
a minus energy c); this reaction will therefore 
occur much more rarely. Activation energies 
are always positive. The total energy change 
for the energetically favorable reaction 
Y —> X, is energy c minus energy b, a 
negative number, which corresponds to 
a loss of free energy. (B) Energy barriers 
for specific reactions can be lowered by 
catalysis, as indicated bythe line marked d. 
Enzymes are particularly effective catalysts 
because they greatly reduce the activation 
energy for the reactions they catalyze. 

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CHAPTER 3 Energy, Catalysis, and Biosynthesis 

Figure 3-13 Lowering the activation 
energy greatly increases the probability 
that a reaction will occur. At any given 
instant, a population of identical substrate 
molecules will have a range of energies, 
distributed as shown on the graph. The 
varying energies come from colllsions with 
surrounding molecules, which make the 
substrate molecules jiggle, vibrate, and 
spin. For a molecule to undergo a Chemical 
reaction, the energy of the molecule must 
exceed the activation energy barrier for that 
reaction (dashed Unes); for most biological 
reactions, this almost never happens 
without enzyme catalysis. Even with enzyme 
catalysis, only a small fraction of substrate 
molecules reach an energy State that is high 
enough forthem to undergo a reaction (red 
shaded area). 

Figure 3-14 Enzymes catalyze reactions 
by lowering the activation energy barrier. 

(A) The dam represents the activation 
energy, which is lowered by enzyme 
catalysis. Each green ball represents 
a potential substrate molecule that is 
bouncing up and down in energy level 
owing to constant encounters with waves, 
an analogy for the thermal bombardment of 
substrate molecules by surrounding water 
molecules. When the barrier—the activation 
energy—is lowered significantly, the balls 
(substrate molecules) with sufficient energy 
can roll downhill, an energetically favorable 
movement. (B) The four walls of the box 
represent the activation energy barriers 
for four different Chemical reactions that 
are all energetically favorable because the 
producís are at lower energy levels than 
the substrates. In the left-hand box, none 
of these reactions occurs because even 
the largest waves are not large enough 
to surmount any of the energy barriers. 

In the rlght-hand box, enzyme catalysis 
lowers the activation energy for reaction 
number 1 only; now the jostling of the waves 
allows the substrate molecule to pass over 
this energy barrier, allowing reaction 1 to 
proceed (Movie 3.1). (C) A branching river 
with a set of barrier dams (yellow boxes) 
serves to ¡Ilústrate how a series of enzyme- 
catalyzed reactions determines the exact 
reaction pathway followed by each molecule 
inside the cell by controlling speciflcally 
which reaction will be allowed at each 

average energy 

activation energy. In the case of a buming book, the activation energy is 
provided by the heat of a lighted match. But cells can't raise their temper- 
ature to drive biological reactions. Inside cells, the push over the energy 
barrier is aided by specialized proteins called enzymes. 

Each enzyme binds tightly to one or two molecules, called substrates, 
and holds them in a way that greatly reduces the activation energy needed 
to facilítate a specific Chemical interaction between them (Figure 3-12B). 
A substance that can lower the activation energy of a reaction is termed 
a catalyst; catalysts increase the rafe of Chemical reactions because they 
allow a much larger proportion of the random collisions with surround¬ 
ing molecules to kick the substrates over the energy barrier, as illustrated 
in Figure 3-13 and Figure 3-14A. Enzymes are among the most effective 
catalysts known. They can speed up reactions by a factor of as much 
as 10 14 (that is, trillions of times faster than the same reactions would 
proceed without an enzyme catalyst). Enzymes therefore allow reactions 
that would not otherwise occur to proceed rapidly at the normal tem- 
perature inside cells. 

LibertadDigital \ 2015 

Free Energy and Catalysis 


r "\ 

molecule A enzyme- enzyme- molecule B 

(substrate) substrate product (product) 

complex complex 

Unlike the effects of temperature, enzymes are highly selective. Each 
enzyme usually speeds up only one particular reaction out of the sev- 
eral possible reactions that its substrate molecules could undergo. In this 
way, enzymes direct each of the many different molecules in a cell along 
specific reaction pathways (Figure 3-14B and C), thereby producing the 
compounds that the cell actually needs. 

Like all catalysts, enzyme molecules themselves remain unchanged after 
participating in a reaction and therefore can function over and over again 
(Figure 3-15). In Chapter 4, we will discuss further how enzymes work, 
after we have looked in detail at the molecular structure of proteins. 

Figure 3-15 Enzymes convert substrates 
to products while remaining unchanged 
themselves. Each enzyme has an active site 
to which one ortwo substrate molecules 
bind, forming an enzyme-substrate 
complex. A reaction occurs at the active 
site, generating an enzyme-product 
complex. The product ¡s then released, 
allowing the enzyme to bind additional 
substrate molecules and repeat the 
reaction. An enzyme thus serves as a 
catalyst, and ¡t usually forms or breaks 
a single covalent bond in a substrate 

The Free-Energy Change for a Reaction Determines 
Whether It Can Occur 

According to the second law of thermodynamics, a Chemical reaction 
can proceed only if it results in a net (overall) increase in the disorder of 
the universe (see Figure 3-5). Disorder increases when useful energy that 
could be hamessed to do work is dissipated as heat. The useful energy in 
a system is known as its free energy, or G. And because Chemical reac¬ 
tions involve a transition frorn one molecular State to another, the term 
that is of most interest to chemists and cell biologists is the free-energy 
change, denoted AG ("Delta G"). 

Let's consider a collection of molecules. AG measures the amount of dis¬ 
order created in the universe when a reaction involving these molecules 
takes place. Energetically favorable reactions, by definition, are those that 
create disorder by decreasing the free energy of the system to which they 
belong; in other words, they have a negativo AG (Figure 3-16). 

A reaction can occur spontaneously only if AG is negative. On a macro- 
scopic scale, an energetically favorable reaction with a negative AG is the 
relaxation of a compressed spring into an expanded State, releasing its 
stored elastic energy as heat to its surroundings. On a microscopic scale, 
an energetically favorable reaction with a negative AG occurs when salt 
(NaCl) dissolves in water. Note that, just because a reaction can occur 
spontaneously, does not mean it will occur quickly. The decay of dia- 
monds into graphite is a spontaneous process—but it takes millions of 

Energetically unfavorable reactions, by contrast, create order in the 
universe; they have a positive AG. Such reactions—for example, the 
formation of a peptide bond between two amino acids—cannot occur 
spontaneously; they take place only when they are coupled to a second 
reaction with a negative AG large enough that the net AG of the entire 
process is negative (Figure 3-17). Life is possible because enzymes can 
create biological order by coupling energetically unfavorable reactions 
with energetically favorable ones. These critical concepts are summa- 
rized, with examples, in Panel 3-1 (pp. 96-97). 





The free energy of Y 
is greater than the free 
energy of X. Therefore 
AG is negative (< 0), and 
the disorder of the 
universe increases 
during the reaction 

this reaction can occur spontaneously 

If the reaction X—«-Y 
occurred, AG would 
be positive (> 0), and 
the universe would 
become more 

this reaction can occur only if 
it is coupled to a second, 
energetically favorable reaction 

Figure 3-16 Energetically favorable 
reactions have a negative AG, whereas 
energetically unfavorable reactions have 
a positive AG. 




LibertadDigital \ 2015 


CHAPTER 3 Energy, Catalysis, and Biosynthesis 

Figure 3-17 Reaction coupling can drive 
an energetically unfavorable reaction. The 

energetically unfavorable (AG > 0) reaction 
X—> Ycannot occur unless it is coupled to 
an energetically favorable (AG < 0) reaction 
C —> D, such that the net free-energy 
change for the coupled reactions is negative 
(less than 0). 


Consider the analogy of the jiggling 
box containing coins that was 
described on page 85. The reaction, 
the flipping of coins that either 
face heads up (H) or tails up (T), is 
described by the equation 
H «-» T, where the rate of the 
forward reaction equals the rate of 
the reverse reaction. 

A. What are AG and AG° in this 

B. What corresponds to the 
temperature at which the reaction 
proceeds? What corresponds to the 
activation energy of the reaction? 
Assume you have an "enzyme," 
called jigglase, which catalyzes this 
reaction. What would the effect of 
jigglase be and what, mechanically, 
might jigglase do in this analogy? 

AG Changes As a Reaction Proceeds Toward Equilibrium 

It's easy to see how a tensed spring, when left to itself, will relax and 
release its stored energy to the environment as heat. But Chemical 
reactions are a bit more complex—and harder to intuit. That’s because 
whether a reaction will proceed depends not only on the energy stored 
in each individual molecule, but also on the concentrations of the mol- 
ecules in the reaction mixture. Recalling our coin analogy, more coins 
in a jiggling box will flip from a head to a tail orientation when the box 
contains 90 heads and 10 tails, than when the box contains 10 heads and 
90 tails. 

The same is true for a Chemical reaction. As the energetically favorable 
reaction Y—> X proceeds, the concentration of the product X will increase 
and the concentration of the substrate Y will decrease. This change in 
relative concentrations of substrate and product will cause the ratio of Y 
to X to shrink, making the initially favorable AG less and less negative. 
Unless more Y is added, the reaction will slow and eventually stop. 
Because AG changes as producís accumulate and substrates are depleted, 
Chemical reactions will generally proceed until they reach a State of 
equilibrium. At that point, the rates of the forward and reverse reactions 
are equal, and there is no further net change in the concentrations of 
substrate or product (Figure 3-18). For reactions at Chemical equilibrium, 
AG = 0, so the reaction will not proceed forward or backward, and no 
work can be done. 

Such a State of Chemical inactivity would be incompatible with life. Living 
cells avoid reaching a State of complete Chemical equilibrium because 
they are constantly exchanging materials with their environment: replen- 
ishing nutrients and eliminating waste products. Many of the individual 
reactions in the cell's complex metabolic network also exist in disequilib- 
rium because the products of one reaction are continually being siphoned 
off to become the substrates in a subsequent reaction. Rarely do products 
and substrates reach concentrations at which the forward and reverse 
reaction rates are equal. 

The Standard Free-Energy Change, AG°, Makes ¡t Possible 
to Compare the Energetics of Different Reactions 

Because AG depends on the concentrations of the molecules in the reac¬ 
tion mixture at any given time, it is not a particularly useful valué for 
comparing the relative energies of different types of reactions. But such 
energetic assessments are necessaiy, for example, to predict whether an 
energetically favorable reaction is likely to have a AG negative enough 
to drive an energetically unfavorable reaction. To compare reactions in 
this way, we need to turn to the standard ftee-energy change of a reaction, 
AG°. The AG° is independent of concentration; it depends only on the 
intrinsic characters of the reacting molecules, based on their behavior 
under ideal conditions where the concentrations of all the reactants are 
set to the same fixed valué of 1 mole/liter. 

A large body of thermodynamic data has been collected from which AG° 
can be calculated for most metabolic reactions. Some common reactions 
are compared in terms of their AG° in Panel 3-1 (pp. 96-97). 

The AG of a reaction can be calculated from AG° if the concentrations of 
the reactants and products are known. For the simple reaction Y —> X, 
their relationship follows this equation: 

AG = AG° + RT ln — 


where AG is in kilocalories per mole, [Y] and [X] denote the concentrations 

LibertadDigital \ 2015 

Free Energy and Catalysis 




¿tík - 

Figure 3-18 Reactions will eventually 
reach a Chemical equilibrium. At that 
point, the forward and the backward 
fluxes of reacting molecules are equal and 
opposite. The widths of the arrows indícate 
the relative rates at which an individual 
molecule converts. 

when X and Y are at equal concentrations, [Y] = [X], the formation of X 
is energetically favored. In other words, the AG of Y —» X is negative and 
the AG of X —► Y is positive. But because of thermal bombardments, 
there will always be some X convertlng to Y. 


O —► 

Y © 

r — -o 

© © 

Therefore the ratio of X to Y 
molecules will increase 

conversión of 
Y to X will 

Conversión of X to Y 
will occur less often 
than the transition 
Y —» X, because it 
requires a more 
energetic collision. 

EVENTUALLY, there will be a large enough excess of X over Y to just 
compénsate for the slow rate of X —> Y, such that the number of Y molecules 
being converted to X molecules each second is exactly equal to the number 
of X molecules being converted to Y molecules each second. At this point, 
the reaction will be at equilibrium. 

v u 

Jl J r? 


AT EQUILIBRIUM, there is no net change in the ratio of Y to X, and the 
AG for both forward and backward reactions is zero. 

of Y and X in moles/liter, ln is the natural logarithm, and RT is the prod- 
uct of the gas constant, R, and the absolute temperature, T. At 37°C, 
RT = 0.616. (A mole is 6 x 10 23 molecules of a substance.) 

From this equation, we can see that when the concentrations of reac- 
tants and producís are equal, in other words, [X]/[Y] = 1, the valué of AG 
equals the valué of AG° (because ln 1 = 0). Thus when the reactants and 
products are present in equal concentrations, the direction of the reac¬ 
tion depends entirely on the intrinsic properties of the molecules. 

The Equilibrium Constant Is Directly Proportional to AG° 

As mentioned earlier, all Chemical reactions tend to proceed toward 
equilibrium. Knowing where that equilibrium lies for any given reaction 
will tell you which way the reaction will proceed—and how far it will 
go. For example, if a reaction is at equilibrium when the concentration 
of the product is ten times the concentration of the substrate, and we 
begin with a surplus of substrate and little or no product, the reaction 
will proceed forward for some time. For the simple reaction Y —> X, that 
valué—the ratio of substrate to product at equilibrium—is called the reac- 
tion's equilibrium constant, K. Expressed as an equation: 

where [X] is the concentration of the product and [Y] is the concentration 
of the substrate at equilibrium. 

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Free Energy and Catalysis 




Energy, Catalysis, and Biosynthesis 







Standard Free Energy 
(AG°) of X minus Free 
Energy of Y in kcal/ 

10 5 


10 4 


10 3 


10 2 




1 0 

10- 1 


10- 2 


10- 3 


10- 4 


10- 5 


Valúes ofthe equilibrium constant were 
calculated for the simple Chemical reaction 
Y <-> X, using the equation given in the text. 

The AG° valúes given here are in kilocalories 
per mole at 37°C. As explained in the text, 

AG° represents the free-energy difference 
under standard conditions (where all 
components are present at a concentraron 
of 1 mole/liter). 

From this table, we see that, ¡f there ¡s a 
favorable free-energy change of 
-4.3 kcal/mole for the transition Y —> X, 
there will be 1000 times more molecules 
ofXthan ofY at equilibrium. 

But how do we know at what concentrations of substrate and product a 
reaction will reach equilibrium? It goes back to the intrinsic properties of 
the molecules involved, as expressed by AG°. Let's see why. 

At equilibrium, the rate of the forward reaction is exactly balanced by 
the rate of the reverse reaction. At that point, AG = 0, and there is no net 
change of free energy to drive the reaction in either direction (see Panel 
3-1, pp. 96-97). 

Now, if we return to the equation presented on p. 94, 

A G = AG° +RT ln — 


we can see that, at equilibrium at 37°C, where AG = 0 and the constant 
RT - 0.616, this equation becomes: 

AG° =-0.616 ln — 


In other words, AG° is directly proportional to the equilibrium constant, K: 
AG° = -0.616 ln K 

If we convert this equation from natural log (ln) to the more commonly 
used base-10 logarithm (log), we get 
AG° = -1.42 log K 

This equation reveáis how the equilibrium ratio of Y to X, expressed as 
the equilibrium constant K, depends on the intrinsic character of the 
molecules, as expressed in the valué of AG° (Table 3-1). It tells us that 
for every 1.42 kcal/mole difference in free energy at 37°C, the equilib¬ 
rium constant changes by a factor of 10. Thus, the more energetically 
favorable the reaction, the more product will accumulate if the reaction 
proceeds to equilibrium. 

ln Complex Reactions, the Equilibrium Constant Ineludes 
the Concentrations of All Reactants and Products 

We have so far discussed the simplest of reactions, Y —► X, in which a 
single substrate is converted into a single product. But inside cells, it is 
more common for two reactants to combine to form a single product: 
A + B ^ AB. How can we predict how this reaction will proceed? 

The same principies apply, except that in this case the equilibrium con¬ 
stant K ineludes the concentrations of both of the reactants, in addition 
to the concentration of the product: 

K= [AB]/[A][B] 

As illustrated in Figure 3-19, the concentrations of both reactants are 
multiplied because the formation of product AB depends on the collision 
of A and B, and these encounters occur at a rate that is proportional to 
[A] x [B], As with single-substrate reactions, AG° = -1.42 log K at 37°C. 

The Equilibrium Constant Indicates the Strength of 
Molecular Interactions 

The concept of free-energy change does not only apply to Chemical reac¬ 
tions where covalent bonds are being broken and formed, but also to 
interactions where one molecule binds to another by means of noncov- 
alent interactions (see Chapter 2, p. 63). Noncovalent interactions are 
immensely important to cells. They inelude the binding of substrates to 
enzymes, the binding of gene regulatory proteins to DNA, and the bind¬ 
ing of one protein to another to make the many different structural and 
functional protein complexes that opérate in a living cell. 

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Free Energy and Catalysis 


concentration concentration 

association rate = k 0 „ [A] [B] 

A B - 

dissociation rate = 


association rate = dissociation rate 
k on [A] [B] = Ac off [AB] 

[AB] k 0 n 

- =- = K = equilibrium constant 

[A] [B] fc off 

Figure 3-19 The equilibrium constant (K) 
for the reaction A + B —> AB depends on 
both the association and dissociation rate 
constants. Molecules A and B must collide 
in orderto interact, and the association 
rate is therefore proportional to the 
product of their individual concentrations 
[A] x [B], As shown, the ratio of the rate 
constants k on and k 0 ff for the association 
and the dissociation reactions, respectively, 
is equal to the equilibrium constant, K, 
for the interaction. Fortwo interacting 
components, klnvolvesthe concentrations 
of both substrates, in addition to that of the 
product. However, the relationship between 
K and AG° is the same as that shown in 
Table 3-1. The larger the valué of K, the 
stronger is the binding between A and B. 

Two molecules will bind to each other if the free-energy change for the 
interaction is negative; that is, the free energy of the resulting complex is 
lower than the sum of the free energies of the two partners when unbound. 
Because the equilibrium constant of a reaction is related directly to AG°, 
K is commonly employed as a measure of the binding strength of a non- 
covalent interaction between two molecules. The binding strength is a 
very useful quantity to know because it also indicates how specific the 
interaction is between the two molecules. 

Consider the reaction that was shown in Figure 3-19, where molecule A 
interacts with molecule B to form the complex AB. The reaction proceeds 
until it reaches equilibrium, at which point the number of association 
events precisely equals the number of dissociation events; at this point, 
the concentrations of reactants A and B, and of the complex AB, can be 
used to determine the equilibrium constant K. 

K becomes larger as the binding energy —that is, the energy released in 
the binding interaction—increases. In other words, the larger K is, the 
greater is the drop in free energy between the dissociated and associ- 
ated States, and the more tightly the two molecules will bind. Even a 
change of a few noncovalent bonds can have a striking effect on a bind¬ 
ing interaction, as illustrated in Figure 3-20. In this example, eliminating 
a few hydrogen bonds from a binding interaction can be seen to cause a 
dramatic decrease in the amount of complex that exists at equilibrium. 

For Sequential Reactions, the Changes in Free Energy Are 

Now we retum to our original concern: how can enzymes catalyze reac¬ 
tions that are energetically unfavorable? One way they do so is by directly 
coupling energetically unfavorable reactions with energetically favorable 
ones. Consider, for example, two sequential reactions, 

X —> Y and Y —► Z 

where the AG° valúes are +5 and -13 kcal/mole, respectively. (Recall that 
a mole is 6 x 10 23 molecules of a substance.) The unfavorable reaction, 
X —y Y, will not occur spontaneously. However, it can be driven by the 
favorable reaction Y —» Z, provided that the second reaction follows 
the first. That's because the overall free-energy change for the coupled 
reaction is equal to the sum of the free-energy changes for each indi¬ 
vidual reaction. In this case, the AG° for the coupled reaction will be 
-8 kcal/mole, making the overall pathway energetically favorable. 

Consider 1000 molecules of A and 1000 
molecules of B in the cytosol of a eukaryotic 
cell. The concentration of both will be 
about 10 -9 M. 

If the equilibrium constant ( K) for 
A + B <-> AB is 10 10 liters/mole, then at 
equilibrium there will be 

270 270 730 


molecules molecules complexes 

If the equilibrium constant is a little weaker, 
say 10 8 liters/mole—a valué that represents a 
loss of 2.8 kcal/mole of binding energy from 
the example above, or 2-3 fewer hydrogen 
bonds—then there will be 

915 915 85 


molecules molecules complexes 

Figure 3-20 Small changes in the 
number of weak bonds can have drastic 
effects on a binding interaction. This 
example illustrates the dramatic effect of 
the presence or absence of a few weak 
noncovalent bonds ¡n the Interaction 
between two cytosollc protelns. 

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100 CHAPTER 3 Energy, Catalysis, and Biosynthesis 


For the reactions shown ¡n Figure 
3-21, sketch an energy diagram 
similar to that in Figure 3-12 for 
the two reactions alone and for 
the combined reactions. Indícate 
the standard free-energy changes 
for the reactions X —► Y, Y —» Z, 
and X —♦ Z in the graph. Indicate 
how enzymes that catalyze these 
reactions would change the energy 

Cells can therefore cause the energetically unfavorable transition, 
X —> Y, to occur if an enzyme catalyzing the X —> Y reaction is supplemented 
by a second enzyme that catalyzes the energetically favorable reaction, 
Y —> Z. In effect, the reaction Y —> Z acts as a "siphon," pulling the conver¬ 
sión of all of molecule X to molecule Y, and then to molecule Z (Figure 
3-21). For example, several of the reactions in the long pathway that 
converts sugars into C0 2 and H 2 0 are energetically unfavorable. The 
pathway nevertheless proceeds rapidly to completion, however, because 
the total AG° for the series of sequential reactions has a large negative 

Forming a sequential pathway, however, is not the answer for all meta- 
bolic needs. Often the desired reaction is simply X —»■ Y, without further 
conversión of Y to some other product. Fortunately, there are other, more 
general ways of using enzymes to couple reactions together, involving 
the production of activated carriers that can shuttle energy from one 
reaction site to another. We discuss these Systems shortly. Before we do, 
let's pause to look at how enzymes find and recognize their substrates 
and how enzyme-catalyzed reactions proceed. After all, thermodynamic 
considerations merely establish whether Chemical reactions can occur; 
enzymes actually make them happen. 

Thermal Motion Allows Enzymes to Find Their Substrates 

Enzymes and their substrates are both present in relatively small amounts 
in the cytosol of a cell, yet a typical enzyme can capture and process 
about a thousand substrate molecules every second. This means that an 
enzyme can release its product and bind a new substrate in a fraction of 
a millisecond. Flow do these molecules find each other so quickly in the 
crowded cytosol of the cell? 

Rapid binding is possible because molecular motions are enormously 
fast. Because of heat energy, molecules are in constant motion and con- 
sequently will explore the cytosolic space very efficiently by wandering 

Figure 3-21 An energetically unfavorable 
reaction can be driven by an energetically 
favorable follow-on reaction that acts 
as a Chemical siphon. (A) At equilibrium, 
there are twice as many X molecules as 

Y molecules. (B) At equilibrium, there are 
25 times more Z molecules than Y 
molecules. (C) If the reactions in (A) and (B) 
are coupled, nearly all of the X molecules 
will be converted to Z molecules, as shown. 
In terms of energetics, the AG° ofthe 

Y —* Z reaction is so negative that, when 
coupled to the X —> Y reaction, it lowers 
the AG of X —> Y, because the AG of X —* Y 
decreases as the ratio of Y to X declines. As 
shown in Figure 3-18, arrow widths reflect 
the relative rates at which an individual 
molecule converts; the arrow lengths are the 
same in both directions here, indicating that 
there is no net flux. 

(C) | equilibrium point for the coupled reaction X —> Y —> Z 

LibertadDigital \ 2015 

Free Energy and Catalysis 


Figure 3-22 A molecule traverses the cytosol by taking a random 
walk. Molecules in solution move in a random fashion due to the 
continual buffeting they receive in collisions with other molecules. This 
movement allows small molecules to diffuse rapldly throughout the 
cell cytosol (Movie 3.2). 

randomly through it—a process called diffusion. In this way, every mol¬ 
ecule in the cytosol collides with a huge number of other molecules each 
second. As the molecules in a liquid collide and bounce off one another, 
an individual molecule moves flrst one way and then another, its path 
constituting a random walk (Figure 3-22). 

Although the cytosol of a cell is densely packed with molecules of vari- 
ous shapes and sizes (Figure 3-23), experiments in which fluorescent 
dyes and other labeled molecules are injected into the cell cytosol show 
that small organic molecules diffuse through this aqueous gel nearly as 
rapidly as they do through water. A small organic molecule, such as a 
substrate, takes only about one-fifth of a second on average to diffuse a 
distance of 10 pm. Diffusion is therefore an efficient way for small mol¬ 
ecules to move limited distances in the cell. 

Because proteins diffuse through the cytosol much more slowly than do 
small molecules, the rate at which an enzyme will encounter its sub¬ 
strate depends on the concentration of the substrate. The most abundant 
substrates are present in the cell at a concentration of about 0.5 mM. 
Because puré water is 55 M, there is only about one such substrate mol¬ 
ecule in the cell for every 10 5 water molecules. Nevertheless, the site on 
an enzyme that binds this substrate will be bombarded by about 500,000 
random collisions with the substrate every second. For a substrate con¬ 
centration tenfold lower (0.05 mM), the number of collisions drops to 
50,000 per second, and so on. 

The random encounters between an enzyme and its substrate often lead 
to the formation of an enzyme-substrate complex. This association is 
stabilized by the formation of múltiple, weak bonds between the enzyme 
and substrate. These weak interactions—which can inelude hydrogen 
bonds, van der Waals attractions, and electrostatic attractions (discussed 
in Chapter 2)—persist until random thermal motion causes the molecules 
to dissociate again. When two colliding molecules have poorly match- 
ing surfaces, few noncovalent bonds are formed, and their total energy 
is negligible compared with that of thermal motion. In this case, the two 
molecules dissociate as rapidly as they come together (see Figure 2-33). 
This is what prevenís incorrect and unwanted associations from form- 
ing between mismatched molecules, such as those between an enzyme 
and the wrong substrate. But when the enzyme and substrate are well 
matched, they form many weak interactions, which keep them held 
together long enough for a covalent bond in the substrate molecule to 
be formed or broken. Knowing the speed at which molecules collide and 
come apart, as well as how fast bonds can be formed and broken, makes 
the observed rate of enzymatic catalysis seem a little less amazing. 

Figure 3-23 The cytosol is crowded with various molecules. Only 
the macromolecules, which are drawn to scale, are shown. RNAs are 
blue, rlbosomes are green, and proteins are red. Enzymes and other 
macromolecules diffuse relatively slowly ¡n the cytosol, in part because 
they ¡nteract with so many other macromolecules. Small molecules, 
by contrast, can diffuse nearly as rapldly as they do ¡n water. (Adapted 
from D.S. Goodsell, Trends Biochem. Sci. 16:203-206, 1991. With 
permission from Elsevier.) 

• _ • 

net distance 


The enzyme carbonic anhydrase 
is one of the speediest enzymes 
known. It catalyzes the rapid 
conversión of CO 2 gas into the 
much more soluble bicarbonate ion 
(HCO 3 - ). The reaction: 

CO 2 + H 2 0 <-► HCO 3 - + H + 
is very important for the efficient 
transport of CO 2 from tissue, where 
CO 2 is produced by respiration, 
to the lungs, where it is exhaled. 
Carbonic anhydrase accelerates the 
reaction 10 7 -fold, hydrating 10 5 CO 2 
molecules per second at its maximal 
speed. What do you suppose limits 
the speed of the enzyme? Sketch 
a diagram analogous to the one 
shown in Figure 3-13 and indícate 
which portion of your diagram has 
been designed to display the 
10 7 -fold acceleration. 

100 nm 

LibertadDigital \ 2015 

102 CHAPTER 3 Energy, Catalysis, and Biosynthesis 

V max and Km Measure Enzyme Performance 

To catalyze a reaction, an enzyme must first bind its substrate. The sub- 
strate then undergoes a reaction to form the product, which initially 
remains bound to the enzyme. Finally, the product is released and dif- 
fuses away, leaving the enzyme free to bind another substrate molecule 
and catalyze another reaction (see Figure 3-15). The rates of the different 
steps vary widely from one enzyme to another, and they can be meas- 
ured by mixing purified enzymes and substrates together under carefully 
defined conditions in a test tube (see How We Know, pp. 104-106). 

In such experiments, the substrate is introduced in increasing concentra- 
tions to a solution containing a fixed concentration of enzyme. At first, 
the concentration of the enzyme-substrate complex—and therefore the 
rate at which product is formed—rises in a linear fashion in direct pro- 
portion to substrate concentration. However, as more and more enzyme 
molecules become occupied by substrate, this rate increase tapers off, 
until at a very high concentration of substrate it reaches a máximum 
valué, termed V max . At this point, the active sites of all enzyme molecules 
in the sample are fully occupied by substrate, and the rate of product for- 
mation depends only on how rapidly the substrate molecule can undergo 
a reaction to form the product. For many enzymes, this tumover number 
is of the order of 1000 substrate molecules per second, although turnover 
numbers between 1 and 100,000 have been measured. 

Because there is no clearly defined substrate concentration at which the 
enzyme can be deemed fully occupied, biochemists instead use a differ¬ 
ent parameter to gauge the concentration of substrate needed to make 
the enzyme work efficiently. This valué is called the Michaelis constant, 
Km, named after one of the biochemists who worked out the relation- 
ship. The Km of an enzyme is defined as the concentration of substrate 
at which the enzyme works at half its máximum speed (Figure 3-24). In 
general, a small Km indicates that a substrate binds very tightly to the 
enzyme, and a large Km indicates weak binding. 

Although an enzyme (or any catalyst) functions to lower the activation 
energy for a reaction such as Y —► X, it is important to note that the 
enzyme will also lower the activation energy for the reverse reaction 
X —► Y to exactly the same degree. The forward and backward reactions 
will therefore be accelerated by the same factor by an enzyme, and the 
equilibrium point for the reaction—and thus its AG°—remains unchanged 
(Figure 3-25). 


In cells, an enzyme catalyzes 
the reaction AB —> A + B. It was 
isolated, however, as an enzyme that 
carries out the opposite reaction 
A + B —» AB. Explain the paradox. 

K m substrate concentration —*- 

Figure 3-24 An enzyme's performance depends on how rapidly it can process 
its substrate. The rate of an enzyme reaction (V) increases as the substrate 
concentration increases, until a máximum valué (V max ) is reached. Atthis point, all 
substrate-binding sites on the enzyme molecules are fully occupied, and the rate 
of the reaction is limited by the rate of the catalytic process on the enzyme surface. 
For most enzymes, the concentration of substrate at which the reaction rate is half- 
maximal (Km) is a direct measure of how tightly the substrate is bound, with a large 
valué of Km (a large amount of substrate needed) corresponding to weak binding. 

LibertadDigital \ 2015 

Activated Carriers and Biosynthesis 


o 0 c c 






The energy released by energetically favorable reactions such as the oxi- 
dation of food molecules must be stored temporarily before it can be 
used by cells to fuel energetically unfavorable reactions, such as the syn- 
thesis of all the other molecules needed by the cell. In most cases, the 
energy is stored as chemical-bond energy in a set of activated carríers, 
small organic molecules that contain one or more energy-rich covalent 
bonds. These molecules diffuse rapidly and carry their bond energy from 
the sites of energy generation to the sites where energy is used for bio¬ 
synthesis or for other energy-requiring cell activities (Figure 3-26). 
Activated carriers store energy in an easily exchangeable form, either 
as a readily transferable Chemical group or as readily transferable ("high 
energy") electrons. They can serve a dual role as a source of both energy 
and Chemical groups for biosynthetic reactions. The most important acti¬ 
vated carriers are ATP and two molecules that are closely related to each 
other, NADH and NADPH. Cells use activated carriers like money to pay 
for the energetically unfavorable reactions that otherwise would not take 

Figure 3-25 Enzymes cannot change 
the equilibrium point for reactions. 

Enzymes, like all catalysts, speed up the 
forward and reverse rates of a reaction by 
the same amount. Therefore, for both the 
(A) uncatalyzed and (B) catalyzed reactions 
shown here, the number of molecules 
undergolng the transltion X —> Y ¡s equal 
to the number of molecules undergolng 
the transition Y —> X when the ratio of 
Y molecules to X molecules ¡s 3.5 to 1, 
as illustrated. In other words, both the 
catalyzed and uncatalyzed reactions will 
eventually reach the same equilibrium point, 
although the catalyzed reaction will reach 
equilibrium faster. 

The Formation of an Activated Carrier Is Coupled to an 
Energetically Favorable Reaction 

When a fuel molecule such as glucose is oxidized in a cell, enzyme-cat- 
alyzed reactions ensure that a large part of the free energy released is 
captured in a chemically useful form, rather than being released waste- 
fully as heat. (Oxidizing sugar in a cell allows you to power metabolic 
reactions, whereas burning a chocolate bar in the Street will get you 
nowhere, producing no metabolically useful energy.) In cells, energy 
capture is achieved by means of a coupled reaction, in which an ener¬ 
getically favorable reaction is used to drive an energetically unfavorable 
one that produces an activated carrier or some other useful molecule. 


Figure 3-26 Activated carriers can store 
and transfer energy in a form that cells 
can use. By serving as ¡ntracellular energy 
shuttles, activated carriers perform their 
function as go-betweens that linkthe 
release of energy from the breakdown of 
food molecules (catabolism) to the energy- 
requiring biosynthesis of small and large 
organic molecules (anabolism). 

LibertadDigital \ 2015 




At first glance, it seems that a cell's metabolic pathways 
have been pretty well mapped out, with each reaction 
proceeding predictably to the next—substrate X is con¬ 
verted to product Y, which is passed along to enzyme Z. 
So why would anyone need to know exactly how tightly 
a particular enzyme clutches its substrate or whether 
it can process 100 or 1000 substrate molecules eveiy 

In reality, such elabórate metabolic maps merely sug- 
gest which pathways a cell might follow as it converts 
nutrients into small molecules, Chemical energy, and the 
larger building blocks of life. Like a road map, they do 
not predict the density of traffic under a particular set 
of conditions: which pathways the cell will use when it 
is starving, when it is well fed, when oxygen is scarce, 
when it is stressed, or when it decides to divide. The 
study of an enzyme's kinetics —how fast it operates, how 
it handles its substrate, how its activity is controlled— 
makes it possible to predict how an individual catalyst 
will perform, and how it will interact with other enzymes 
in a network. Such knowledge leads to a deeper under- 
standing of cell biology, and it opens the door to leaming 
how to harness enzymes to perform desired reactions. 


The first step to understanding how an enzyme performs 
involves determining the maximal velocity, Vmax, for the 
reaction it catalyzes. This is accomplished by measur- 
ing, in a test tube, how rapidly the reaction proceeds 

in the presence of different concentrations of substrate 
(Figure 3-27A): the rate should increase as the amount 
of substrate rises until the reaction reaches its Vmax- The 
velocity of the reaction is measured by monitoring either 
how quickly the substrate is consumed or how rapidly 
the product accumulates. In many cases, the appear- 
ance of product or the disappearance of substrate can be 
observed directly with a spectrophotometer. This instru- 
ment detects the presence of molecules that absorb light 
at a particular wavelength; NADH, for example, absorbs 
light at 340 nm, while its oxidized counterpart, NAD + , 
does not. So, a reaction that generates NADH (by reduc- 
ing NAD + ) can be monitored by following the formation 
of NADH at 340 nm in a spectrophotometer. 

To determine the Vmax of a reaction, you would set up a 
series of test tubes, where each tube contains a different 
concentration of substrate. For each tube, add the same 
amount of enzyme and then measure the velocity of the 
reaction—the number of micromoles of substrate con¬ 
sumed or product generated per minute. Because these 
numbers will tend to decrease over time, the rate used is 
the velocity measured early in the reaction. These initial 
velocity valúes (v) are then plotted against the substrate 
concentration, yielding a curve like the one shown in 
Figure 3-27B. 

Looking at this plot, however, it is difficult to deter¬ 
mine the exact valué of V max , as it is not clear where 
the reaction rate will reach its plateau. To get around 
this problem, the data are converted to their reciprocáis 

Figure 3-27 Measured reaction rates are plotted to determine V max and Km of an enzyme-catalyzed reaction. (A) A series 
of increasing substrate concentrations is prepared, a fixed amount of enzyme is added, and initial reaction rates (velocities) are 
determined. (B) The initial velocities (v) plotted against the substrate concentrations [S] give a curve described by the general equation 
y = ax/(b + x). Substituting our kinetic terms, the equation becomes v = V max [S]/(KM + [S]), where V max is the asymptote of the curve (the 
valué of y at an infinite valué of x), and Km ¡s equal to the substrate concentration where v is one-half V max . This is called the Michaelis- 
Menten equation, named for the biochemists who provided evidence for this enzymatic relationship. (C) In a double-reciprocal plot, 1/v 
is plotted against 1/[S], The equation describing this straight line is 1/v = (KM/V max )(1/[S]) + 1/V max . When 1/[S] = 0, the y ¡ntercept (1/v) 
is 1/V max . When 1/v= 0, the x ¡ntercept (1/[S]) is -1/Km. Plotting the data this way allows V max and Km to be calculated more precisely. By 
convention, lowercase letters are used for variables (henee vfor velocity) and uppercase letters are used for constants (henee V max ). 

íibertadDigital 1 2015 


and graphed in a "double-reciprocal plot," where the 
inverse of the velocity (1/v) appears on the y axis and 
the inverse of the substrate concentration (1/[SJ) on the 
x axis (Figure 3-27C). This graph yields a straight line 
whose y intercept (the point where the line crosses the 
y axis) represents 1 /V max and whose x intercept corre- 
sponds to -1/Km- These valúes are then converted to 
valúes for V max and Km- 

Enzymologists use this technique to determine the 
kinetic parameters of many enzyme-catalyzed reactions 
(although these days Computer programs automatically 
plot the data and spit out the sought-after valúes). Some 
reactions, however, happen too fast to be monitored 
in this way; the reaction is essentially complete—the 
substrate entirely consumed—within thousandths of a 
second. For these reactions, a special piece of equip- 
ment must be used to follow what happens during the 
first few milliseconds after enzyme and substrate meet 
(Figure 3-28). 


Substrates are not the only molecules that can influ- 
ence how well or how quickly an enzyme works. In 
many cases, producís, substrate lookalikes, inhibitors, 
and other small molecules can also increase or decrease 
enzyme activity. Such regulation allows cells to control 
when and how rapidly various reactions occur, a proc- 
ess we will consider in more detail in Chapter 4. 

Determining how an inhibitor decreases an enzyme's 
activity can reveal how a metabolic pathway is regu- 
lated—and can suggest how those control points can be 
circumvented by carefully designed mutations in specific 

The effect of an inhibitor on an enzyme's activity is mon¬ 
itored in the same way that we measured the enzyme's 
kinetics. A curve is first generated showing the velocity 
of the uninhibited reaction between enzyme and sub¬ 
strate, as described previously. Additional curves are 
then produced for reactions in which the inhibitor mol- 
ecule has been included in the mix. 

Comparing these curves, with and without inhibitor, can 
also reveal how a particular inhibitor impedes enzyme 
activity. For example, some inhibitors bind to the same 
site on an enzyme as its substrate. These competitive 
inhibitors block enzyme activity by competing directly 
with the substrate for the enzyme's attention. They 
resemble the substrate enough to tie up the enzyme, 
but they differ enough in structure to avoid getting con¬ 
verted to product. This blockage can be overeóme by 
adding enough substrate so that enzymes are more 
likely to encounter a substrate molecule than an inhib¬ 
itor molecule. From the kinetic data, we can see that 
competitive inhibitors do not change the Vmax of a reac¬ 
tion; in other words, add enough substrate and the 
enzyme will encounter mostly substrate molecules and 
will reach its máximum velocity (Figure 3-29). 

light source 


Figure 3-28 A stopped-flow apparatus is used to observe reactions during the first few milliseconds. In this piece of equipment, 
the enzyme and substrate are rapidly injected into a mixing chamber through two syringes. The enzyme and its substrate meet as they 
shoot through the mixing tube at flow rates that can easily reach 1000 cm/sec. They then enter another tube and zoom past a detector 
that monitors, say, the appearance of product. If the detector is located within a centimeter of where the enzyme and substrate meet, ¡t 
is possible to observe reactions when they are only a few milliseconds oíd. 

LibertadDigital \ 2015 


e . ¡ir iu 

•v X* 

•© » 






Figure 3-29 A competitive inhibitor directly blocks 
substrate binding to an enzyme. (A) The active site of 
the enzyme can bind either the competitive inhibitor or 
the substrate, but not both together. (B) The upper piot 
shows that inhibition by a competitive inhibitor can be 
overeóme by increasing the substrate concentration. 

The double-reciprocal plot below shows that the V max 
of the reaction is not changed in the presence of the 
competitive inhibitor: the y intercept is ¡dentical for both 
the curves. 

Competitive inhibitors can be used to treat patients who 
have been poisoned by ethylene glycol, an ingredient in 
commercially available antifreeze. Although ethylene 
glycol is itself not fatally toxic, a by-product of its metab- 
olism—oxalic acid—can be lethal. To prevent oxalic acid 
from forming, the patient is given a large (though not 
quite intoxicating) dose of ethanol. Ethanol competes 
with the ethylene glycol for binding to alcohol dehydro- 
genase, the first enzyme in the pathway to oxalic acid 
formation. As a result, the ethylene glycol goes mostly 
unmetabolized and is safely eliminated from the body. 
Other types of inhibitors may interact with sites on the 
enzyme distant from where the substrate binds. As we 
discuss in Chapter 4, many biosynthetic enzymes are 
regulated by feedback inhibition, whereby an enzyme 
early in a pathway will be shut down by a product gener- 
ated later in the pathway. Because this type of inhibitor 
binds to a sepárate regulatory site on the enzyme, the 
substrate can still bind, but it might do so more slowly 
than it would in the absence of inhibitor. Such noncom- 
petitive inhibition is not overeóme by the addition of 
more substrate. 


With the kinetic data in hand, we can use Computer 
modeling programs to predict how an enzyme will per- 
form, and even how a cell will respond when exposed 
to different conditions—such as the addition of a par¬ 
ticular sugar or amino acid to the culture médium, or 
the addition of a poison or a pollutant. Seeing how a 
cell manages its resources—which pathways it favors 
for dealing with particular biochemical challenges—can 
also suggest strategies for designing better catalysts for 
reactions of medical or commercial importance (e.g., 
for producing drugs or detoxifying industrial waste). 
Using such tactics, bacteria have even been genetically 

engineered to produce large amounts of indigo—the 
dye, originally extracted from plants, that makes your 
blue jeans blue. 

Computer programs have been developed to facilítate 
the dissection of complex reaction pathways. They 
require information about the components in the path¬ 
way, including the Km and V max of the participating 
enzymes and the concentrations of enzymes, substrates, 
produets, inhibitors, and other regulatory molecules. 
The program then prediets how molecules will flow 
through the pathway, which produets will be gener- 
ated, and where any bottlenecks might be. The process 
is not unlike balancing an algebraic equation, in which 
every atom of carbón, nitrogen, oxygen, and so on must 
be tallied. Such careful accounting makes it possible 
to rationally design ways to manipúlate the pathway, 
such as re-routing it around a bottleneck, eliminating 
an important inhibitor, redirecting the reactions to favor 
the generation of predominantly one product, or extend- 
ing the pathway to produce a novel molecule. Of course, 
such Computer models must be validated in cells, which 
may not always behave as predicted. 

Producing designer cells that spew out commercial 
produets generally requires using genetic engineering 
techniques to introduce the gene or genes of choice into 
a cell, usually a bacterium, that can be manipulated and 
maintained in the laboratory. We discuss these methods 
at greater length in Chapter 10. Hamessing the power of 
cell biology for commercial purposes—even to produce 
something as simple as the amino acid tiyptophan—is 
currently a multibillion-dollar industiy. And, as more 
genome data come in, presenting us with more enzymes 
to exploit, it may not be long before vats of custom-made 
bacteria are churning out drugs and Chemicals that rep- 
resent the biological equivalent of puré gold. 

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Activated Carriers and Biosynthesis 


Figure 3-30 A mechanical model illustrates the principie of coupled Chemical reactions. The spontaneous 
reaction shown ¡n (A) could serve as an analogy for the direct oxidation of glucose to CO2 and H2O, which produces 
only heat. In (B), the same reaction ¡s coupled to a second reaction, which could serve as an analogy for the synthesis 
of activated carriers. The energy produced in (B) ¡s in a more useful form than in (A) and can be used to drive a 
variety of otherwise energetically unfavorable reactions (C). 

Such coupling requires enzymes, which are fundamental to all of the 
energy transactions in the cell. 

The nature of a coupled reaction is illustrated by a mechanical analogy 
in Figure 3-30, in which an energetically favorable Chemical reaction 
is represented by rocks falling frorn a cliff. The kinetic energy of falling 
rocks would normally be entirely wasted in the form of heat generated by 
friction when the rocks hit the ground (Figure 3-30A). By careful design, 
however, part of this energy could be used to drive a paddle wheel that 
lifts a bucket of water (Figure 3-30B). Because the rocks can now reach 
the ground only after moving the paddle wheel, we say that the energeti¬ 
cally favorable reaction of rocks falling has been directly coupled to the 
energetically unfavorable reaction of lifting the bucket of water. Because 
part of the energy is used to do work in (B), the rocks hit the ground with 
less velocity than in (A), and correspondingly less energy is wasted as 
heat. The energy saved in the elevated bucket of water can then be used 
to do useful work (Figure 3-30C). 

Analogous processes occur in cells, where enzymes play the role of the 
paddle wheel in Figure 3-30B. By mechanisms that we discuss in Chapter 
13, enzymes couple an energetically favorable reaction, such as the oxi¬ 
dation of foodstuffs, to an energetically unfavorable reaction, such as the 
generation of activated carriers. As a result, the amount of heat released 
by the oxidation reaction is reduced by exactly the amount of energy that 
is stored in the energy-rich covalent bonds of the activated carrier. That 
saved energy can then be used to power a Chemical reaction elsewhere 
in the cell. 

ATP Is the Most Widely Used Activated Carrier 

The most important and versatile of the activated carriers in cells is ATP 
(adenosine 5'-triphosphate). Just as the energy stored in the raised bucket 
of water in Figure 3-30B can be used to drive a wide variety of hydraulic 
machines, ATP serves as a convenient and versatile store, or currency, of 
energy that can be used to drive a variety of Chemical reactions in cells. 


Use Figure 3-30B to ¡Ilústrate the 
following reaction driven by the 
hydrolysis of ATP: 

X + ATP —► Y + ADP + P¡ 

A. In this case, which molecule or 
molecules would be analogous to 
(i) rocks at top of cliff, (ii) broken 
debris at bottom of cliff, (iii) bucket 
at its highest point, and (iv) bucket 
on the ground? 

B. What would be analogous to 
(i) the rocks hitting the ground in 
the absence of the paddle wheel in 
Figure 3-30A and (ii) the hydraulic 
machine in Figure 3-30C? 

LibertadDigital \ 2015 

108 CHAPTER 3 Energy, Catalysis, and Biosynthesis 

Figure 3-31 The interconversion of 
ATP and ADP occurs in a cycle. The two 

outermost phosphate groups in ATP are 
held to the rest of the molecule by high- 
energy phosphoanhydride bonds and 
are readily transferred to other organic 
molecules. Water can be added to ATP to 
form ADP and ¡norganic phosphate (P¡). 
Inside a cell, this hydrolysis of the terminal 
phosphate of ATP yields between 11 and 
13 kcal/mole of usable energy. Although 
the AG° of this reaction ¡s -7.3 kcal/mole, 
the AG is much more negative, because the 
ratio of ATP to the products ADP and P¡ ¡s 
so high inside the cell. 

The large negative AG° of the reaction 
arises from a number of factors. Release of 
the terminal phosphate group removes an 
unfavorable repulsión between adjacent 
negative charges; in addition, the ¡norganic 
phosphate ion (P¡) released is stabilized by 
favorable hydrogen-bond formation with 
water. The formation of ATP from ADP and 
P¡ reverses the hydrolysis reaction; because 
this condensaron reaction is energetically 
unfavorable, it must be coupled to an 
energetically more favorable reaction to 

phosphoanhydride bonds 

energy available 
to drive energetically 

As shown in Figure 3-31 , ATP is synthesized in an energetically unfa¬ 
vorable phosphoiylation reaction, in which a phosphate group is added 
to ADP (adenosine 5'-diphosphate). When required, ATP gives up this 
energy packet in an energetically favorable hydrolysis to ADP and inor- 
ganic phosphate (P¡). The regenerated ADP is then available to be used 
for another round of the phosphorylation reaction that forms ATP, creat- 
ing an ATP cycle in the cell. 

Figure 3-32 The terminal phosphate 
of ATP can be readily transferred to 
other molecules. Because an energy- 
rich phosphoanhydride bond in ATP 
is converted to a less energy-rich 
phosphoester bond in the phosphate- 
accepting molecule, this reaction is 
energetically favorable, having a large 
negative AG° (see Panel 3-1, pp. 96-97). 
Phosphorylation reactions ofthis type are 
involved in the synthesis of phospholipids 
and in the initial steps of the breakdown of 
sugars, as well as in many other metabolic 
and ¡ntracellular slgnaling pathways. 

The energetically favorable reaction of ATP hydrolysis is coupled to 
many otherwise unfavorable reactions through which other molecules 
are synthesized. We will encounter several of these reactions in this 
chapter, where we will see exactly how this is done. ATP hydrolysis is 
often coupled to the transfer of the terminal phosphate in ATP to another 
molecule, as illustrated in Figure 3-32. Any reaction that involves the 
transfer of a phosphate group to a molecule is termed a phosphorylation 
reaction. Phosphorylation reactions are examples of condensation reac¬ 
tions (see Figure 2-25), and they occur in many important cell processes: 
they actívate substrates, medíate the exchange of Chemical energy, and 
serve as key constituents of intracellular signaling pathways (discussed 
in Chapter 16). 


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Activated Carriers and Biosynthesis 


ATP is the most abundant activated carrier in cells. It is used, for example, 
to supply energy for many of the pumps that actively transport sub- 
stances into or out of the cell (discussed in Chapter 12); it also powers the 
molecular motors that enable muscle cells to contract and nerve cells to 
transport materials along their lengthy axons (discussed in Chapter 17). 
Why evolution selected this particular nucleotide over the others as the 
major carrier of energy, however, remains a mystery. The nucleotide GTP, 
although similar, has very different functions in the cell, as we discuss in 
later chapters. 

Energy Stored in ATP Is Often Harnessed to Join Two 
Molecules Together 

A common type of reaction that is needed for biosynthesis is one in which 
two molecules, A and B, are joined together by a covalent bond to pro¬ 
duce A-B in the energetically unfavorable condensation reaction: 

A-H + B-OH -> A-B + H 2 0 

ATP hydrolysis can be coupled indirectly to this reaction to make it go 
forward. In this case, energy frorn ATP hydrolysis is first used to con- 
vert B-OH to a higher-energy intermedíate compound, which then reacts 
directly with A-H to give A-B. The simplest mechanism involves the 
transfer of a phosphate frorn ATP to B-OH to make B-O-PO 3 , in which 
case the reaction pathway contains only two steps: 

1. B-OH + ATP -► B-O-PO3 + ADP 

2. A-H + B-O-PO3 -» A-B + P¡ 

Net result: B-OH + ATP + A-H A-B + ADP + P¡ 

The condensation reaction, which by itself is energetically unfavora¬ 
ble, has been forced to occur by being coupled to ATP hydrolysis in an 
enzyme-catalyzed reaction pathway (Figure 3-33A). 

A biosynthetic reaction of exactly this type is employed to synthesize the 
amino acid glutamine, as illustrated in Figure 3-33B. We will see later in 
the chapter that very similar (but more complex) mechanisms are also 
used to produce nearly all of the large molecules of the cell. 


The phosphoanhydride bond that 
links two phosphate groups in 
ATP in a high-energy linkage has a 
AG° of-7.3 kcal/mole. Hydrolysis 
of this bond in a cell liberates from 
11 to 13 kcal/mole of usable energy. 
How can this be? Why do you think 
a range of energies is given, rather 
than a precise number as for AG°? 

NADH and NADPH Are Both Activated Carriers of 

Other important activated carriers particípate in oxidation-reduction 
reactions and are commonly part of coupled reactions in cells. These 
activated carriers are specialized to carry both high-energy electrons and 
hydrogen atoms. The most important of these electrón carriers are NADH 
(nicotinamide adenine dinucleotide) and the closely related molecule 
NADPH (nicotinamide adenine dinucleotide phosphate). Both NADH and 
NADPH carry energy in the form of two high-energy electrons plus a pro¬ 
tón (H + ), which together form a hydride ion (H - ). When these activated 
carriers pass their energy (in the form of a hydride ion) to a donor mol¬ 
ecule, they become oxidized to form NAD+ and NADP+, respectively. 

Like ATP, NADPH is an activated carrier that participates in many 
important biosynthetic reactions that would otherwise be energeti¬ 
cally unfavorable. NADPH is produced according to the general scheme 
shown in Figure 3-34A. During a special set of energy-yielding catabolic 
reactions, a hydride ion is removed from the substrate molecule and 
added to the nicotinamide ring of NADP+ to form NADPH. This is a typi- 
cal oxidation-reduction reaction; the substrate is oxidized and NADP + is 

LibertadDigital \ 2015 

110 CHAPTER 3 Energy, Catalysis, and Biosynthesis 





high-energy intermedíate 




+ I 

H 3 N + —CH —COO 
high-energy intermedíate 

Figure 3-33 An energetically unfavorable biosynthetic reaction can be driven by ATP hydrolysis. 

(A) Schematic ¡Ilustraron of the formation of A-B in the condensaron reaction described ¡n the text. (B) The 
biosynthesis of the amino acid glutamine from glutamic acid. Glutamic acid is first converted to a high-energy 
phosphorylated intermedíate (corresponding to the compound B-O-PO3 described in the text), which then reacts 
with ammonia (corresponding to A-H) to form glutamine. In this example, both steps occur on the surface of the 
same enzyme, glutamine synthetase (not shown). For clarity, the glutamic acid side chain is shown in its uncharged 
form. ATP hydrolysis can drive this energetically unfavorable reaction because it yields more energy (AG° of 
-7.3 kcal/mole) than the energy required for the synthesis of glutamine from glutamic acid plus NH3 (AG° 
of +3.4 kcal/mole). 

The hydride ion carried by NADPH is given up readily in a subsequent 
oxidation-reduction reaction, because the ring can achieve a more stable 
arrangement of electrons without it. In this subsequent reaction, which 
regenerates NADP+, the NADPH becomes oxidized and the substrate 
becomes reduced—thus completing the NADPH cycle. NADPH is efficient 
at donating its hydride ion to other molecules for the same reason that 
ATP readily transfers a phosphate: in both cases, the transfer is accompa- 
nied by a large negative free-energy change. One example of the use of 
NADPH in biosynthesis is shown in Figure 3-35. 

NADPH and NADH Have Different Roles ¡n Cells 

NADPH and NADH differ in a single phosphate group, which is located far 
from the región involved in electrón transfer in NADPH (Figure 3-34B). 
Although this phosphate group has no effect on the electron-transfer 
properties of NADPH compared with NADH, it is nonetheless crucial 
for their distinctive roles, as it gives NADPH a slightly different shape 
from NADH. This subtle difference in conformation makes it possible for 
the two carriers to bind as substrates to different sets of enzymes and 
thereby deliver electrons (in the form of hydride ions) to different target 

Why should there be this división of labor? The answer lies in the need 
to regúlate two sets of electron-transfer reactions independently. NADPH 
operates chiefly with enzymes that catalyze anabolic reactions, supply- 
ing the high-energy electrons needed to synthesize energy-rich biological 
molecules. NADH, by contrast, has a special role as an intermedíate in 

LibertadDigital \ 2015 

—.. NADP + i 

NADP + o 



the catabolic system of reactions that generate ATP through the oxida- 
tion of food molecules, as we discuss in Chapter 13. The génesis of NADH 
from NAD + and that of NADPH from NADP+ occurs by different pathways 
that are independently regulated, so that the cell can adjust the supply of 
electrons for these two contrasting purposes. Inside the cell, the ratio of 
NAD + to NADH is kept high, whereas the ratio of NADP+ to NADPH is kept 
low. This arrangement provides plenty of NAD + to act as an oxidizing 
agent and plenty of NADPH to act as a reducing agent—as required for 
their special roles in catabolism and anabolism, respectively. 

Cells Make Use of Many Other Activated Carriers 

In addition to ATP (which transfers a phosphate) and NADPH and NADH 
(which transfer electrons and hydrogen), cells make use of other activated 
carriers that pick up and carry a Chemical group in an easily transferred, 
high-energy linkage. FADH 2 , like NADH and NADPH, carries hydrogen and 
high-energy electrons (see Figure 13-13B). But other important reactions 
involve the transfers of acetyl, methyl, carboxyl, and glucose groups from 
activated carriers for the purpose of biosynthesis (Table 3-2). Coenzyme 
A, for example, can carry an acetyl group in a readily transferable link¬ 
age. This activated carrier, called acetyl CoA (acetyl coenzyme A), is 
shown in Figure 3-36. It is used, for example, to add sequentially two- 
carbon units in the biosynthesis of the hydrocarbon tails of fatty acids. 

Activated Carriers and Biosynthesis 

Figure 3-34 NADPH is an 
activated carrier of electrons. 

(A) NADPH is produced in 
reactions of the general type 
shown on the left, in which two 
hydrogen atoms are removed from 
a substrate. The oxidized form 
of the carrier molecule, NADP + , 
receives one hydrogen atom plus 
an electrón (a hydride ¡on), while 
the protón (H + ) from the other 
H atom is released into solution. 
Because NADPH holds its hydride 
¡on in a high-energy linkage, the 
¡on can easily be transferred to 
other molecules, as shown on the 
right. (B) The structure of NADP + 
and NADPH. On the left is a ball- 
and-stick model of NADP. The part 
ofthe NADP + molecule known as 
the nicotinamide ring accepts two 
electrons, together with a protón 
(the equivalent of a hydride ion, 
H“), forming NADPH. NAD+ and 
NADH are identical in structure to 
NADP + and NADPH, respectively, 
except that they lack the 
phosphate group, as indicated. 


Figure 3-35 NADPH participates ir 
final stage of one of the biosynthetic 
routes leading to cholesterol. As in many 
other biosynthetic reactions, the reduction 
ofthe C=C bond is achieved by the transfer 
of a hydride ¡on from the activated carrier 
NADPH, plus a protón (H + ) from solution. 

LibertadDigital \ 2015 



Energy, Catalysis, and Biosynthesis 


1 Activated Carrier 

1 Group Carried in High-Energy Linkage 1 




electrons and hydrogens 

Acetyl CoA 

acetyl group 

Carboxylated biotin 

carboxyl group 


methyl group 

Uridine diphosphate glucose 


In acetyl CoA and the other activated carriers in Table 3-2, the transfer- 
able group makes up only a small part of the molecule. The rest consists 
of a large organic portion that serves as a convenient "handle," facilitat- 
ing the recognition of the carrier molecule by specific enzymes. As with 
acetyl CoA, this handle portion veiy often contains a núcleotide. This 
curious fact may be a relie from an early stage of cell evolution. It is 
thought that the main catalysts for early life forms on Earth were RNA 
molecules (or their cióse relatives) and that proteins were a later evo- 
lutionaiy addition. It is therefore tempting to speculate that many of the 
activated carriers that we find today originated in an earlier RNA world, 
where their nucleotide portions would have been useful for binding these 
carriers to RNA-based catalysts, or ribozymes (discussed in Chapter 7). 
Activated carriers are usually generated in reactions coupled to ATP 
hydrolysis, as shown for biotin in Figure 3-37. Therefore, the energy that 
enables their groups to be used for biosynthesis ultimately comes from 
the catabolic reactions that generate ATP. Similar processes occur in the 
synthesis of the very large macromolecules—the nucleic acids, proteins, 
and polysaccharides—which we discuss next. 

Figure 3-36 Acetyl coenzyme A (CoA) is 
another important activated carrier. 

A ball-and-stick model is shown above the 
structure of acetyl CoA. The sulfur atom 
(yellow) forms a thioester bond to acétate. 
Because the thioester bond is a high- 
energy linkage, it releases a large amount 
of free energy when it is hydrolyzed; thus 
the acetyl group carried by CoA can be 
readily transferred to other molecules. 

LibertadDigital \ 2015 

Activated Carriers and Biosynthesis 



pyruvate carboxylase oxaloacetate 


Figure 3-37 An activated carrier transfers a carboxyl group to a substrate. Biotin ¡s a vitamin that is used by 
a number of enzymes, ¡ncluding pyruvate carboxylase shown here. Once it is carboxylated, biotin can transfer a 
carboxyl group to another molecule. Here, it transfers a carboxyl group to pyruvate, producing oxaloacetate, a 
molecule needed in the citric acid cycle (discussed in Chapter 13). Other enzymes use biotin to transfer carboxyl 
groups to other acceptor molecules. Note that the synthesis of carboxylated biotin requires energy derived from ATP 
hydrolysis—a general feature of many activated carriers. 

The Synthesis of Biological Polymers Requires an Energy 

The macromolecules of the cell constitute the vast majority of its diy 
mass—that is, the mass not due to water. These molecules are made 
from subunits (or monomers) that are linked together by bonds formed 
during an enzyme-catalyzed condensation reaction. The reverse reac- 
tion—the breakdown of polymers—occurs through enzyme-catalyzed 
hydrolysis reactions. These hydrolysis reactions are energetically favora¬ 
ble, whereas the corresponding biosynthetic reactions require an energy 
input and are more complex (Figure 3-38). 

The nucleic acids (DNA and RNA), proteins, and polysaccharides are all 
polymers that are produced by the repeated addition of a subunit onto 
one end of a growing chain. The mode of synthesis of each of these 
macromolecules is outlined in Figure 3-39. As indicated, the condensa¬ 
tion step in each case depends on energy provided by the hydrolysis of a 
nucleoside triphosphate. And yet, except for the nucleic acids, there are 
no phosphate groups left in the final product molecules. How, then, is the 
energy of ATP hydrolysis coupled to polymer synthesis? 

ho- B 

h 2 o 





h 2 o 





A-H + HO- B 

Figure 3-38 In cells, macromolecules are 
synthesized by condensation reactions 
and broken down by hydrolysis reactions. 

Condensation reactions are all energetically 
unfavorable, whereas hydrolysis reactions 
are all energetically favorable. 

LibertadDigital \ 2015 

114 CHAPTER 3 Energy, Catalysis, and Biosynthesis 


Which of the following reactions will 
occur only if coupled to a second, 
energetically favorable reaction? 

A. glucose + O2 —► CO2 + H2O 

B. CO2 + H2O —► glucose + O2 

C. nucleoside triphosphates —» DNA 

D. nucleotide bases —> nucleoside 

E. ADP + P¡ -► ATP 

For each type of macromolecule, an enzyme-catalyzed pathway exists, 
which resembles that discussed previously for the synthesis of the amino 
acid glutamine (see Figure 3-33). The principie is exactly the same, in 
that the -OH group that will be removed in the condensation reaction is 
first activated by forming a high-energy linkage to a second molecule. 
The mechanisms used to link ATP hydrolysis to the synthesis of proteins 
and polysaccharides, however, are more complex than that used for 
glutamine synthesis. In the biosynthetic pathways leading to these mac- 
romolecules, a series of high-energy intermediates generates the final 
high-energy bond that is broken during the condensation step (as dis¬ 
cussed in Chapter 7 for protein synthesis). 

There are limits to what each activated carrier can do in driving biosyn¬ 
thesis. For example, the AG for the hydrolysis of ATP to ADP and inorganic 
phosphate (P¡) depends on the concentrations of all of the reactants, and 
under the usual conditions in a cell, is between -11 and -13 kcal/mole. 
In principie, this hydrolysis reaction can be used to drive an unfavorable 
reaction with a AG of, perhaps, +10 kcal/mole, provided that a suitable 
reaction path is available. For some biosynthetic reactions, however, 
even -13 kcal/mole may be insufficient. In these cases, the path of ATP 

LibertadDigital \ 2015 

Activated Carriers and Biosynthesis 


adenosine triphosphate (ATP) 

O O 

-O-P-OH + O P Olí 

¿- ¿r 

phosphate phosphate 


Figure 3-40 In an alternative route for 
the hydrolysis of ATP, pyrophosphate 
is first formed and then hydrolyzed in 
solution. This route releases about twice 
as much free energy as the reaction shown 
earlier ¡n Figure 3-31. (A) In each of the two 
successive hydrolysis reactions, an oxygen 
atom from the participating water molecule 
is retained in the producís, whereas the 
hydrogen atoms from water form free 
hydrogen ions, H + . (B) The overall reaction 
shown in summary form. 

(D@ + mi 

h 2 o—> 

® + ® 

hydrolysis can be altered so that it initially produces AMP and pyrophos¬ 
phate (PP¡), which is itself then hydrolyzed in solution in a subsequent 
step (Figure 3-40). The whole process makes available a total AG of 
about -26 kcal/mole. The biosynthetic reaction involved in the synthesis 
of nucleic acids (polynucleotides) is driven in this way (Figure 3-41). 

ATP will make many appearances throughout the book as a molecule 
that powers reactions in the cell. And in Chapters 13 and 14, we discuss 
how the cell uses the energy from food to generate ATP. In the next chap- 
ter, we learn more about the proteins that make such reactions possible. 

Figure 3-41 Synthesis of a polynucleotide, 
RNA or DNA, is a multistep process 
driven by ATP hydrolysis. In the first step, 
a nucleoside monophosphate is activated 
by the sequential transfer of the terminal 
phosphate groups from two ATP molecules. 
The high-energy intermedíate formed—a 
nucleoside triphosphate—exists free in 
solution until it reacts with the growing 
end of an RNA or a DNA chain with release 
of pyrophosphate. Hydrolysis ofthe 
pyrophosphate to inorganic phosphate 
is highly favorable and helps to drive 
the overall reaction in the direction of 
polynucleotide synthesis. 

LibertadDigital \ 2015 

116 CHAPTER 3 Energy, Catalysis, and Biosynthesis 


• Living organisms are able to exist because of a continual input of 
energy. Part of this energy is used to carry out essential reactions 
that support cell metabolism, growth, movement, and reproduction; 
the remainder is lost in the form of heat. 

• The ultimate source of energy for most living organisms is the sun. 
Plants, algae, and photosynthetic bacteria use solar energy to pro¬ 
duce organic molecules from carbón dioxide. Animáis obtain food by 
eating plants or by eating animáis that feed on plants. 

• Each of the many hundreds of Chemical reactions that occur in a cell 
is specifically catalyzed by an enzyme. Large numbers of different 
enzymes work in sequence to form chains of reactions, called meta- 
bolic pathways, each performing a different function in the cell. 

• Catabolic reactions release energy by breaking down organic mol¬ 
ecules, including foods, through oxidative pathways. Anabolic 
reactions generate the many complex organic molecules needed by 
the cell, and they require an energy input. In animal cells, both the 
building blocks and the energy required for the anabolic reactions are 
obtained through catabolic reactions. 

• Enzymes catalyze reactions by binding to particular substrate mol¬ 
ecules in a way that lowers the activation energy required for making 
and breaking specific covalent bonds. 

• The rate at which an enzyme catalyzes a reaction depends on how 
rapidly it finds its substrates and how quickly the product forms and 
then diffuses away. These rates vaiy widely from one enzyme to 

• The only Chemical reactions possible are those that increase the 
total amount of disorder in the universe. The free-energy change for 
a reaction, A G, measures this disorder, and it must be less than zero 
for a reaction to proceed spontaneously. 

• The AG for a Chemical reaction depends on the concentrations of the 
reacting molecules, and it may be calculated from these concentra¬ 
tions if the equilibrium constant ( K) of the reaction (or the standard 
free-energy change, AG°, for the reactants) is known. 

• Equilibrium constants govem all of the associations (and dissocia- 
tions) that occur between macromolecules and small molecules in 
the cell. The larger the binding energy between two molecules, the 
larger the equilibrium constant and the more likely that these mol¬ 
ecules will be found bound to each other. 

• By creating a reaction pathway that couples an energetically favora¬ 
ble reaction to an energetically unfavorable one, enzymes can make 
otherwise impossible Chemical transformations occur. 

• A small set of activated carriers, particularly ATP, NADH, and NADPH, 
plays a central part in these coupled reactions in cells. ATP carries 
high-energy phosphate groups, whereas NADH and NADPH carry 
high-energy electrons. 

• Food molecules provide the carbón skeletons for the formation of 
macromolecules. The covalent bonds of these larger molecules are 
produced by condensation reactions that are coupled to energeti¬ 
cally favorable bond changes in activated carriers such as ATP and 

LibertadDigital \ 2015 

Chapter 3 End-of-Chapter Questions 



acetyl CoA 

free energy, G 

activated carrier 

free-energy change, AG 

activation energy 







Michaelis constant (Km) 







condensation reaction 


coupled reaction 





standard free-energy change, AG° 




turnover number 

equilibrium constant, K 




Which of the following statements are corred? Explain your 

A. Some enzyme-catalyzed readions cease completely ¡f 
their enzyme is absent. 

B. High-energy eledrons (such as those found in the 
adivated carriers NADH and NADPH) move faster around 
the atomic nucleus. 

C. Hydrolysis of ATP to AMP can provide about twice as 
much energy as hydrolysis of ATP to ADP. 

D. A partially oxidized carbón atom has a somewhat smaller 
diameter than a more reduced one. 

E. Some adivated carrier molecules can transfer both 
energy and a chemical group to a second molecule. 

F. The rule that oxidations release energy, whereas 
redudions require energy input, applies to all chemical 
readions, not just those that occur in living cells. 

G. Cold-blooded animáis have an energetic disadvantage 
because they release less heat to the environment than 
warm-blooded animáis do. This slows their ability to make 
ordered macromolecules. 

H. Linking the readion X —> Y to a second, energetically 
favorable readion Y —» Z will shift the equilibrium constant 
of the first readion. 


Consider a transition of X —> Y. Assume that the only 
difference between X and Y is the presence of three 
hydrogen bonds in Y that are absent in X. What is the ratio 
of X to Y when the readion is in equilibrium? Approximate 
your answer by using Table 3-1 (p. 98), with 1 kcal/mole 
as the energy of each hydrogen bond. If Y instead has 
six hydrogen bonds that distinguish it from X, how would 
that change the ratio? 


Protein A binds to protein B to form a complex, AB. 

At equilibrium in a cell the concentrations of A, B, and AB 
are all at 1 pM. 

A. Referring to Figure 3-19, calcúlate the equilibrium 
constant for the readion A + B ^ AB. 

B. What would the equilibrium constant be if A, B, and 
AB were each present in equilibrium at the much lower 
concentrations of 1 nM each? 

C. How many extra hydrogen bonds would be needed to 
hold A and B together at this lower concentration so that 
a similar proportion of the molecules are found in the AB 
complex? (Remember that each hydrogen bond contributes 
about 1 kcal/mole.) 

LibertadDigital \ 2015 

118 CHAPTER 3 Energy, Catalysis, and Biosynthesis 


Discuss the following statement: "Whether the AG for a 
reaction is larger, smaller, or the same as AG° depends on 
the concentration of the compounds that particípate in the 


A. How many ATP molecules could maximally be generated 
from one molecule of glucose, if the complete oxidation of 

1 mole of glucose to CO2 and H2O yields 686 kcal of free 
energy and the useful Chemical energy available in the high- 
energy phosphate bond of 1 mole of ATP is 12 kcal? 

B. As we will see in Chapter 14 (Table 14-1), respiration 
produces 30 moles of ATP from 1 mole of glucose. Compare 
this number with your answer in part (A). What is the overall 
efficiency of ATP production from glucose? 

C. If the cells of your body oxidize 1 mole of glucose, by 
how much would the temperature of your body (assume 
that your body consists of 75 kg of water) increase if the 
heat were not dissipated into the environment? [Recall that 
a kilocalorie (kcal) is defined as that amount of energy that 
heats 1 kg of water by 1 °C.] 

D. What would the consequences be if the cells of your 
body could convert the energy in food substances with 
only 20% efficiency? Would your body—as it is presently 
constructed—work just fine, overheat, or freeze? 

E. A resting human hydrolyzes about 40 kg of ATP every 
24 hours. The oxidation of how much glucose would 
produce this amount of energy? (Hint: Look up the structure 
of ATP in Figure 2-24 to calcúlate its molecular weight; the 
atomic weights of H, C, N, O, and P are 1, 12, 14, 16, and 
31, respectively.) 


A prominent scientist claims to have isolated mutant cells 
that can convert 1 molecule of glucose into 57 molecules 
of ATP. Should this discovery be celebrated, or do you 
suppose that something might be wrong with it? Explain 
your answer. 


In a simple reaction A ^ A*, a molecule is interconvertible 
between two forms that differ in standard free energy G° by 
4.3 kcal/mole, with A* having the higher G°. 

A. Use Table 3-1 (p. 98) to find how many more molecules 
will be in State A* compared with State A at equilibrium. 

B. If an enzyme lowered the activation energy of the 
reaction by 2.8 kcal/mole, how would the ratio of A to A* 


A reaction in a single-step biosynthetic pathway that 
converts a metabolite into a particularly vicious poison 
(metabolite ^ poison) in a mushroom is energetically 
highly unfavorable. The reaction is normally driven by ATP 
hydrolysis. Assume that a mutation in the enzyme that 
catalyzes the reaction prevents it from utilizing ATP, but still 
allows it to catalyze the reaction. 

A. Do you suppose ¡t might be safe for you to eat a 
mushroom that bears this mutation? Base your answer on an 
estimation of how much less poison the mutant mushroom 
would produce, assuming the reaction is in equilibrium 

and most of the energy stored in ATP is used to drive the 
unfavorable reaction in nonmutant mushrooms. 

B. Would your answer be different for another mutant 
mushroom whose enzyme couples the reaction to ATP 
hydrolysis but works 100 times more slowly? 


Consider the effects of two enzymes, A and B. Enzyme A 
catalyzes the reaction 

and enzyme B catalyzes the reaction 

Discuss whether the enzymes would be beneficial or 
detrimental to cells. 


Discuss the following statement: "Enzymes and heat are 
alike in that both can speed up reactions that—although 
thermodynamically feasible—do not occur at an appreciable 
rate because they require a high activation energy. Diseases 
that seem to benefit from the careful application of heat—in 
the form of hot chicken soup, for example—are therefore 
likely to be due to the insufficient function of an enzyme." 


The curve shown in Figure 3-24 is described by the 
Michaelis-Menten equation: 
rate (v) = \/ max [S]/([S] + K M ) 

Can you convince yourself that the features qualitatively 
described in the text are accurately represented by this 
equation? In particular, how can the equation be simplified 
when the substrate concentration [S] is in one of the 
following ranges: (A) [S] is much smaller than the Km, 

(B) [S] equals the K M , and (C) [S] is much larger than the K M ? 


The rate of a simple enzyme reaction is given by the 
standard Michaelis-Menten equation: 
rate = \/ max [S]/([S] + K M ) 

If the V max of an enzyme is 100 pmole/sec and the Km is 
1 mM, at what substrate concentration is the rate 
50 pmole/sec? Plot a graph of rate versus substrate (S) 
concentration for [S] = 0 to 10 mM. Convert this to a plot of 
1/rate versus 1/[S]. Why is the latter plot a straight line? 


Select the correct options in the following and explain your 
choices. If [S] is much smaller than Km, the active site of the 
enzyme is mostly occupied/unoccupied. If [S] is very much 
greater than Km, the reaction rate is limited by the enzyme/ 
substrate concentration. 

LibertadDigital \ 2015 

Chapter 3 End-of-Chapter Questions 119 


A. The reaction rates of the reaction S —» P catalyzed by 
enzyme E were determined under conditions such that only 
very little product was formed. The following data were 

Substrate Reaction rate 

concentration (fxM) ((xmole/min) 

0.08 0.15 

0.12 0.21 

0.54 0.7 

1.23 1.1 

1.82 1.3 

2.72 1.5 

4.94 1.7 

10.00 1.8 

Plot the above data as a graph. Use this graph to estímate 
the Km and the V max for this enzyme. 

B. Recall from the How We Know essay (pp. 104—106) that 
to determine these valúes more precisely, a trick is generally 
used in which the Michaelis-Menten equation is transformed 
so that it is possible to plot the data as a straight line. 

A simple rearrangement yields 

1/rate = (K M /V m ax) (1/[S]) + 1/V max 
which is an equation of the form y = ax + b. Calcúlate 
1/rate and 1/[S] for the data given in part (A) and then plot 
1/rate versus 1/[S] as a new graph. Determine Km and V max 
from the intercept of the line with the axis, where 1/[S] = 0, 
combined with the slope of the line. Do your results agree 
with the estimates made from the first graph of the raw 

C. It is stated in part (A) that only very little product 
was formed under the reaction conditions. Why is this 

D. Assume the enzyme is regulated such that upon 
phosphorylation its Km increases by a factor of 3 without 
changing its \/ max . Is this an activation or inhibition? Plot the 
data you would expect for the phosphorylated enzyme in 
both the graph for (A) and the graph for (B). 

LibertadDigital \ 2015 

left intentionally blank 

LibertadDigital \ 2015 



Protein Structure and Function 

When we look at a cell in a microscope or analyze its electrical or bio- 
chemical activity, we are, in essence, observing the handiwork ofproteins. 
Proteins are the main building blocks from which cells are assembled, 
and they constitute most of the cell’s dry mass. In addition to provid- 
ing the cell with shape and structure, proteins also execute nearly all its 
myriad functions. Enzymes promote intracellular Chemical reactions by 
providing intricate molecular surfaces, contoured with particular bumps 
and crevices that can eradle or exelude specific molecules. Proteins 
embedded in the plasma membrane form the channels and pumps that 
control the passage of nutrients and other small molecules into and out 
of the cell. Other proteins carry messages from one cell to another, or 
act as signal integrators that relay information from the plasma mem¬ 
brane to the nucleus of individual cells. Some proteins act as motors that 
propel organelles through the cytoplasm, and others function as compo- 
nents of tiny molecular machines with precisely calibrated moving parís. 
Specialized proteins also act as antibodies, toxins, hormones, antifreeze 
molecules, elastic fibers, or luminescence generators. Before we can 
hope to understand how genes work, how muscles contract, how nerves 
conduct electricity, how embryos develop, or how our bodies function, 
we musí understand proteins. 

The multiplicity of functions carried out by proteins (Panel 4-1, p. 122) 
arises from the huge number of different shapes they adopt. We therefore 
begin our description of these remarkable macromolecules by discussing 
their three-dimensional structures and the properties that these struc- 
tures confer. We next look at how proteins work: how enzymes catalyze 
Chemical reactions, how some proteins act as molecular switches, and 
how others generate orderly movement. We then examine how cells 





LibertadDigital \ 2015 



The Shape and Structure of Proteins 


control the activity and location of the proteins they contain. Finally, we 
present a brief description of the techniques that biologists use to work 
with proteins, including methods for purifying them—from tissues or cul- 
tured cells—and for determining their structures. 


From a Chemical point of view, proteins are by far the most structur- 
ally complex and functionally sophisticated molecules known. This is 
perhaps not surprising, considering that the structure and activity of 
each protein has been developed and fine-tuned over billions of years 
of evolution. We start by considering how the position of each amino 
acid in the long string of amino acids that forms a protein determines its 
three-dimensional shape, which is stabilized by noncovalent interactions 
between different parts of the molecule. Understanding the structure of a 
protein at the atomic level allows us to see how the precise shape of the 
protein determines its function. 

The Shape of a Protein Is Specified by Its Amino Acid 

Proteins, as you may recall from Chapter 2, are assembled mainly from 
a set of 20 different amino acids, each with different Chemical proper- 
ties. A protein molecule is made from a long chain of these amino acids, 
held together by covalent peptide bonds (Figure 4-1 ). Proteins are there- 
fore referred to as polypeptides, and their amino acid chains are called 
polypeptide chains. In each type of protein, the amino acids are present 
in a unique order, called the amino acid sequence, which is exactly the 
same from one molecule of that protein to the next. One molecule of 
human insulin, for example, has the same amino acid sequence as every 
other molecule of human insulin. Many thousands of different proteins 
have been identified, each with its own distinct amino acid sequence. 

peptide bond in glycylalanine 

Figure 4-1 Amino acids are linked 
together by peptide bonds. A covalent 
peptide bond forms when the carbón atom 
of the carboxyl group of one amino acid 
(such as glycine) shares electrons with the 
nitrogen atom (b/ue) from the amino group 
of a second amino acid (such as alanine). 
Because a molecule of water is eliminated, 
peptide bond formation is classified as a 
condensaron reaction (see Figure 2-29). 

In this diagram, carbón atoms are gray, 
nitrogen b/ue, oxygen red, and 
hydrogen white. 

LibertadDigital \ 2015 


CHAPTER 4 Protein Structure and Function 

Figure 4-2 A protein ¡s made 
of amino acids linked together 
into a polypeptide chain. The 

amino acids are linked by peptide 
bonds (see Figure 4-1) to form 
a polypeptide backbone of 
repeating structure (gray boxes), 
from which the side chain of each 
amino acid projects. The character 
and sequence of the chemically 
distinct side chains—for example, 
nonpolar ( green ), polar uncharged 
(yellow), and negative ( blue ) side 
chains—give each protein its 
distinct, individual properties. 

A small polypeptide of just four 
amino acids ¡s shown here. Proteins 
are typically made up of chains of 
several hundred amino acids, whose 
sequence ¡s always presented 
starting with the N-terminus reading 
from left to right. 

carboxyl terminus 



Aspartic acid 





Each polypeptide chain consists of a backbone that is adorned with a 
variety of Chemical side chains. This polypeptide backbone is formed 
from a repeating sequence of the core atoms (-N-C-C-) found in every 
amino acid (see Figure 4-1). Because the two ends of each amino acid 
are chemically different—one sports an amino group (NH 3 +, also written 
NH 2 ) and the other a carboxyl group (COCT, also written COOH)—each 
polypeptide chain has a directionality: the end carrying the amino group 
is called the amino terminus, or N-terminus, and the end carrying the 
free carboxyl group is the carboxyl terminus, or C-terminus. 

Projecting from the polypeptide backbone are the amino acid side 
chains—the part of the amino acid that is not involved in forming pep¬ 
tide bonds (Figure 4-2). The side chains give each amino acid its unique 
properties: some are nonpolar and hydrophobic ("water-fearing"), some 
are negatively or positively charged, some can be chemically reactive, 
and so on. The atomic formula for each of the 20 amino acids in proteins 
is presented in Panel 2-5 (pp. 74-75), and a brief list of the 20 common 
amino acids, with their abbreviations, is provided in Figure 4-3. 


Aspartic acid 



negatively charged 

Glutamic acid 



negatively charged 




positively charged 




positively charged 




positively charged 




uncharged polar 




uncharged polar 




uncharged polar 




uncharged polar 




uncharged polar 











































Figure 4-3 Twenty different amino acids are commonly found in proteins. Both three-letter and one-letter abbreviations are given, 
as well as the character of the side chain. There are equal numbers of polar (hydrophillc) and nonpolar (hydrophobic) side chains, and 
half of the polar side chains carry a positive or negative charge. 

LibertadDigital \ 2015 

The Shape and Structure of Proteins 


glutamic acid 

Long polypeptide chains are very flexible, as many of the peptide bonds 
that link the carbón atoms in the polypeptide backbone allow free rota- 
tion of the atoms they join. Thus, proteins can in principie fold in an 
enormous number of ways. The shape of each of these folded chains, 
however, is constrained by many sets of weak noncovalent bonds that 
form within proteins. These bonds involve atoms in the polypeptide 
backbone, as well as atoms in the amino acid side chains. The noncova¬ 
lent bonds that help proteins fold up and maintain their shape inelude 
hydrogen bonds, electrostatic attractions, and van der Waals attractions, 
which are described in Chapter 2 (see Panel 2-7, pp. 78-79). Because a 
noncovalent bond is much weaker than a covalent bond, it takes many 
noncovalent bonds to hold two regions of a polypeptide chain tightly 
together. The stability of each folded shape is largely influenced by the 
combined strength of large numbers of noncovalent bonds (Figure 4-4). 

Figure 4-4 Three types of noncovalent 
bonds help proteins fold. Although a 
single one of any of these bonds is quite 
weak, many of them together can create a 
strong bonding arrangement that stabilizes 
a particular three-dimensional structure, 
as in the small polypeptide shown in 
the center. R is often used as a general 
designation for an amino acid side chain. 
Protein folding is also aided by hydrophobic 
forces, as shown in Figure 4-5. 

A fourth weak forcé, hydrophobic interaction, also has a central role in 
determining the shape of a protein. In an aqueous environment, hydro¬ 
phobic molecules, including the nonpolar side chains of particular amino 
acids, tend to be forced together to minimize their disruptive effect on 
the hydrogen-bonded network of the surrounding water molecules (see 
Panel 2-2, pp. 68-69). Therefore, an important factor goveming the fold¬ 
ing of any protein is the distribution of its polar and nonpolar amino 
acids. The nonpolar (hydrophobic) side chains—which belong to amino 
acids such as phenylalanine, leucine, valine, and tryptophan (see Figure 
4-3)—tend to cluster in the interior of the folded protein (just as hydro¬ 
phobic oil droplets coalesce to form one large drop). Tucked away inside 
the folded protein, hydrophobic side chains can avoid contact with the 
aqueous cytosol that surrounds them inside a cell. In contrast, polar side 
chains—such as those belonging to arginine, glutamine, and histidine— 
tend to arrange themselves near the outside of the folded protein, where 
they can form hydrogen bonds with water and with other polar mole¬ 
cules (Figure 4-5). When polar amino acids are buried within the protein, 
they are usually hydrogen-bonded to other polar amino acids or to the 
polypeptide backbone (Figure 4-6). 

LibertadDigital \ 2015 


CHAPTER 4 Protein Structure and Function 

Figure 4-5 Hydrophobic forces help 
proteins fold into compact conformations. 

Polar amino acid side chains tend to be 
displayed on the outside of the folded 
protein, where they can ¡nteract with water; 
the nonpolar amino acid side chains are 
buried on the inside to form a highly packed 
hydrophobic core of atoms that are hidden 
from water. 

unfolded polypeptide 

folded conformation in aqueous environment 

Proteins Fold into a Conformation of Lowest Energy 

Each type of protein has a particular three-dimensional structure, which 
is determined by the order of the amino acids in its polypeptide chain. 
The final folded structure, or conformation, adopted by any polypeptide 
chain is determined by energetic considerations: a protein generally folds 
into the shape in which its free energy (G) is minimized. The folding proc- 
ess is thus energetically favorable, as it releases heat and increases the 
disorder of the universe (see Panel 3-1, pp. 96-97). 

Protein folding has been studied in the laboratoiy using highly purified 
proteins. A protein can be unfolded, or denatured, by treatment with sol- 
vents that disrupt the noncovalent interactions holding the folded chain 
together. This treatment converts the protein into a flexible polypeptide 
chain that has lost its natural shape. Under the right conditions, when the 

Figure 4-6 Hydrogen bonds within a 
protein molecule help stabilize its folded 
shape. Large numbers of hydrogen bonds 
form between adjacent regions of the 
folded polypeptide chain. The structure 
shown is a portion of the enzyme lysozyme. 
Hydrogen bonds between backbone 
atoms are shown in red; those between 
the backbone and a side chain are shown 
in yellow; and those between atoms of two 
side chains are shown in blue. Note that 
the same amino acid side chain can make 
múltiple hydrogen bonds (red arrow). The 
atoms are colored as in Figure 4—1, although 
the hydrogen atoms are not shown. (After 
C.K. Mathews, K.E. van Holde, and 
K.G. Ahern, Biochemistry, 3rd ed. San 
Francisco: Benjamín Cummings, 2000.) 

backbone to backbone 

hydrogen bond between 
atoms of two peptide 

backbone to side chain 

hydrogen bond between 
atoms of a peptide bond 
and an amino acid side chain 

side chain to side chain 

hydrogen bond between 
two amino acid side 

LibertadDigital \ 2015 

The Shape and Structure of Proteins 


denatured protein 

Figure 4-7 Denatured proteins can 
often recover their natural shapes. This 
type of experiment demonstrates that the 
conformation of a protein is determined 
solely by ¡ts amino acid sequence. 
Renaturation requires the corred conditions 
and works best for small proteins. 

denaturing solvent is removed, the protein often refolds spontaneously 
into its original conformation—aprocess called renaturation (Figure 4-7). 
The fact that a denatured protein can, on its own, refold into the cor¬ 
red conformation indicates that all the information necessaiy to specify 
the three-dimensional shape of a protein is contained in its amino acid 

Each protein normally folds into a single stable conformation. This con¬ 
formation, however, often changes slightly when the protein interacts 
with other molecules in the cell. This change in shape is crucial to the 
function of the protein, as we discuss later. 

When proteins fold incorrectly, they sometimes form aggregates that can 
damage cells and even whole tissues. Misfolded proteins are thought 
to contribute to a number of neurodegenerative disorders, such as 
Alzheimer's disease and Huntington's disease. Some infectious neuro¬ 
degenerative diseases—including scrapie in sheep, bovine spongiform 
encephalopathy (BSE, or "mad cow" disease) in cattle, and Creutzfeldt- 
Jakob disease (CJD) in humans—are caused by misfolded proteins called 
prions. The misfolded prion form of a protein can convert the properly 
folded versión of the protein in an infected brain into the abnormal confor¬ 
mation. This allows the misfolded prions, which tend to form aggregates, 
to spread rapidly from cell to cell, eventually causing the death of the 
affected animal or human (Figure 4-8). Prions are considered "infectious" 
because they can also spread from an affected individual to a normal 
individual via contaminated food, blood, or surgical instruments, for 

Although a protein chain can fold into its correct conformation without 
outside help, protein folding in a living cell is generally assisted by spe- 
cial proteins called chaperone proteins. Some of these chaperones bind 
to partly folded chains and help them to fold along the most energeti- 
cally favorable pathway (Figure 4-9). Others form "isolation chambers" 
in which single polypeptide chains can fold without the risk of forming 
aggregates in the crowded conditions of the cytoplasm (Figure 4-10). In 
either case, the final three-dimensional shape of the protein is still speci- 
fied by its amino acid sequence; chaperones merely make the folding 
process more efficient and reliable. 

Proteins Come in a Wide Variety of Complicated Shapes 

Proteins are the most structurally diverse macromolecules in the cell. 
Although they range in size from about 30 amino acids to more than 

Figure 4-8 Prion diseases are caused by proteins whose misfolding 
is infectious. (A) The protein undergoes a rare conformational change 
to give an abnormally folded prion form. (B) The abnormal form causes 
the conversión of normal proteins in the host's brain into a misfolded 
prion form. (C) The prions aggregate into amyloid fibrils, which disrupt 
brain cell function, causing a neurodegenerative disorder, such as 
"mad cow" disease (see also Figure 4-18). 


Urea used in the experiment shown 
in Figure 4-7 is a molecule that 
disrupts the hydrogen-bonded 
network of water molecules. Why 
might high concentrations of urea 
unfold proteins? The structure of 
urea is shown here. 

/ \ 

h 2 n nh 2 

(A) normal protein can, on occasion, adopt 
an abnormal, misfolded prion form 

normal abnormal prion form 

protein of protein 

(B) the prion form of the protein can bind 
tothe normal form, inducing conversión 
to the abnormal conformation 


( conversión of normal 
protein to abnormal 
prion form 

(C) abnormal prion proteins propágate 
and aggregate to form amyloid fibrils 

amyloid fibril 

LibertadDigital \ 2015 


CHAPTER 4 Protein Structure and Function 

Figure 4-9 Chaperone proteins can 
guide the folding of a newly synthesized 
polypeptide chain. The chaperones bind to 
newly synthesized or partially folded chains 
and helping them to fold along the most 
energetically favorable pathway. 

Assoclation of these chaperones with the 
target protein requlres an ¡nput of energy 
from ATP hydrolysis. 

newly synthesized, 
partially folded protein 

/ proteins 

é>d & 

tp & 

10,000, the vast majority are between 50 and 2000 amino acids long. 
Proteins can be globular or fibrous, and they can form filaments, sheets, 
rings, or spheres (Figure 4-11). We will encounter many of these struc- 
tures later in this chapter and throughout the book. 

To date, the structures of about 100,000 different proteins have been 
determined. We discuss how scientists unravel these structures later in 
the chapter. Most proteins have a three-dimensional conformation so 
intricate and irregular that their structure would require an entire chap¬ 
ter to describe in detail. But we can get some sense of the intricacies of 
polypeptide structure by looking at the conformation of a relatively small 
protein, such as the bacterial transport protein HPr. 

This small protein is only 88 amino acids long, and it serves as a car- 
rier protein that facilitates the transport of sugar into bacterial cells. In 
Figure 4-12, we present HPr's three-dimensional structure in four dif¬ 
ferent ways, each of which emphasizes different features of the protein. 
The backbone model (Figure 4-12A) shows the overall organization of 
the polypeptide chain and provides a straightforward way to compare the 
structures of related proteins. The ribbon model (Figure 4-12B) shows the 
polypeptide backbone in a way that emphasizes its various folds, which 
we describe in detail shortly. The wire model (Figure 4-12C) ineludes the 
positions of all the amino acid side chains; this view is especially useful 

newly synthesized, cnan 

partially folded proteins cap 

&>&<% /y 

le polypeptide 
chain is sequestered 
by the chaperone 

chain folds 

correctly folded 
protein is released 
when cap 

Figure 4-10 Other chaperone proteins act as ¡solation chambers that help a 
polypeptide fold. In this case, the barrel of the chaperone provides an enclosed 
chamber in which a newly synthesized polypeptide chain can fold without the risk of 
aggregating with other polypeptides in the crowded conditions of the cytoplasm. 
This System also requires an ¡nput of energy from ATP hydrolysis, mainly for the 
association and subsequent dissociation of the cap that closes off the chamber. 

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The Shape and Structure of Proteins 


Figure 4-11 Proteins come in a variety of shapes and sizes. Each folded polypeptide is shown as a space-filling model, represented 
at the same scale. In the top-left córner is HPr, the small protein featured in detail ¡n Figure 4-12. For comparison we also show a 
portion of a DNA molecule (gray) bound to the protein deoxyribonuclease. (After David S. Goodsell, Our Molecular Nature. New York: 
Sprlnger-Verlag, 1996. Wlth permisslon from Springer Science and Business Media.) 

LibertadDigital \ 2015 


CHAPTER 4 Protein Structure and Function 

(A) backbone model 


(D) space-filling model 

Figure 4-12 Protein conformation can be represented ¡n a variety 
of ways. Shown here ¡s the structure of the small bacterial transport 
protein HPr. The images are colored to make it easier to trace the path 
ofthe polypeptide chain. In these models, the región of polypeptide 
chain carrying the protein's N-terminus is purple and that near its 
C-terminus is red. 

for predicting which amino acids might be involved in the protein's activ- 
ity. Finally, the space-fllling model (Figure 4-12D) provides a contour map 
of the protein surface, which reveáis which amino acids are exposed on 
the surface and shows how the protein might look to a small molecule 
such as water or to another macromolecule in the cell. 

The structures of larger proteins—or of multiprotein complexes—are 
even more complex. To visualize such detailed and complicated struc¬ 
tures, scientists have developed various graphical and computer-based 
tools that generate a variety of images of a protein, only some of which 
are depicted in Figure 4-12. These images can be displayed on a Com¬ 
puter screen and readily rotated and magnified to view all aspects of the 
structure (Movie 4.1). 

When the three-dimensional structures of many different protein 
molecules are compared, it becomes clear that, although the overall con¬ 
formation of each protein is unique, some regular folding patterns can be 
detected, as we discuss next. 

The a Helix and the p Sheet Are Common Folding Patterns 

More than 60 years ago, scientists studying hair and silk discovered two 
common folding patterns present in many different proteins. The flrst to 
be discovered, called the a helix, was found in the protein a-keratin, which 
is abundant in skin and its derivatives—such as hair, nails, and homs. 
Within a year of the discovery of the a helix, a second folded structure, 
called a p sheet, was found in the protein Jibroin, the major constituent 
of silk. (Biologists often use Greek letters to ñame their discoveries, with 
the first example receiving the designation a, the second p, and so on.) 
These two folding patterns are particularly common because they result 
from hydrogen bonds that form between the N-H and C=0 groups in 
the polypeptide backbone (see Figure 4-6). Because the amino acid side 
chains are not involved in forming these hydrogen bonds, a hélices and p 
sheets can be generated by many different amino acid sequences. In each 
case, the protein chain adopts a regular, repeating form. These structural 
features, and the shorthand cartoon symbols that are often used to repre- 
sent them in models of protein structures, are presented in Figure 4-13. 

Hélices Form Readily in Biological Structures 

The abundance of hélices in proteins is, in a way, not surprising. A helix 
is a regular structure that resembles a spiral staircase. It is generated 
simply by placing many similar subunits next to one another, each in 
the same strictly repeated relationship to the one before. Because it is 
veiy rare for subunits to join up in a straight line, this arrangement will 
generally result in a helix (Figure 4-14). Depending on the twist of the 
staircase, a helix is said to be either right-handed or left-handed (Figure 
4-14E). Flandedness is not affected by tuming the helix upside down, but 
it is reversed if the helix is reflected in a mirror. 

An a helix is generated when a single polypeptide chain turns around 
itself to form a structurally rigid cylinder. A hydrogen bond is made 
between every fourth amino acid, linking the C=Q of one peptide bond to 

LibertadDigital \ 2015 

The Shape and Structure of Proteins 


a helix 

. carbón 






the N-H of another (see Figure 4-13A). This gives rise to a regular right- 
handed helix with a complete turn eveiy 3.6 amino acids (Movie 4.2). 
Short regions of a helix are especially abundant in proteins that are 
embedded in cell membranes, such as transport proteins and receptors. 
We will see in Chapter 11 that those portions of a transmembrane pro- 
tein that cross the lipid bilayer usually form an a helix that is composed 
largely of amino acids with nonpolar side chains. The polypeptide back- 
bone, which is hydrophilic, is hydrogen-bonded to itself in the a helix, 
and it is shielded from the hydrophobic lipid environment of the mem- 
brane by its protruding nonpolar side chains (Figure 4-15). 

Sometimes two (or three) a hélices will wrap around one another to 
form a particularly stable structure known as a coiled-coil. This struc¬ 
ture forms when the a hélices have most of their nonpolar (hydrophobic) 
side chains on one side, so that they can twist around each other with 

Figure 4-13 Polypeptide chains often fold 
into one of two orderly repeating forms 
known as an a helix and a P sheet. 

(A-C) In an a helix, the N-H of every 
peptide bond is hydrogen-bonded to 
the C=0 of a neighboring peptide bond 
located four amino acids away in the same 
chain. (D-F) In a P sheet, several segments 
(strands) oían individual polypeptide chain 
are held together by hydrogen-bonding 
between peptide bonds in adjacent strands. 
The amino acid side chains in each strand 
project alternately above and below 
the plañe of the sheet. In the example 
shown, the adjacent chains run in opposite 
directions, forming an antiparallel sheet. 
(A) and (D) show all of the atoms in the 
polypeptide backbone, but the amino acid 
side chains are denoted by R. (B) and (E) 
show only the carbón (black and gray) and 
nitrogen (b/ue) backbone atoms, while (C) 
and (F) display the cartoon symbols that 
are used to represent the a helix and the 
P sheet in ribbon models of proteins (see 
Figure 4-12B). 


Remembering that the amino 
acid side chains projecting from 
each polypeptide backbone in a 
P sheet point alternately above 
and below the plañe of the sheet 
(see Figure 4-13D), consider 
the following protein sequence: 
Leu-Lys-lle-Arg-Phe-Glu. Do you 
find anything remarkable about the 
arrangement of the amino acids in 
this sequence when incorporated 
into a P sheet? Can you make any 
predictions as to how the P sheet 
might be arranged in a protein? 
(Hint: consult the properties of the 
amino acids listed in Figure 4-3.) 

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CHAPTER 4 Protein Structure and Function 

Figure 4-14 The helix is a common, 
regular, biological structure. A helix will 
form when a series of similar subunits 
bind to each other ¡n a regular way. At 
the bottom, the interaction between two 
subunits is shown; behind them are the 
hélices that result. These hélices have two 
(A), three (B), or six (C and D) subunits per 
helical turn. Atthe top, the arrangement 
of subunits has been photographed from 
directly above the helix. Note that the 
helix in (D) has a wider path than that ¡n 
(C), but the same number of subunits per 
turn. (E) A helix can be either right-handed 
or left-handed. As a reference, ¡t is useful 
to remember that standard metal screws, 
which advance when turned clockwise, are 
right-handed. So to judge the handedness 
of a helix, imagine screwing ¡t into a wall. 
Note that a helix preserves the same 
handedness when it is turned upside down. 

hydrophobic amino acid 
side chain 

in lipid bilayer 

Figure 4-15 Many membrane-bound 
proteins cross the lipid bilayer as an 
a helix. The hydrophobic side chains 
of the amino acids forming the a helix 
contad the hydrophobic hydrocarbon 
tails of the phospholipid molecules, while 
the hydrophilic parts of the polypeptide 
backbone form hydrogen bonds with one 
another in the interior of the helix. About 
20 amino acids are required to span a 
membrane in this way. Note that, despite 
the appearance of a space along the interior 
of the helix in this schematic diagram, the 
helix is not a channel: no ions or small 
molecules can pass through it. 

(A) (B) (C) (D) (E) 

these side chains facing inward—minimizing their contact with the aque- 
ous cytosol (Figure 4-16). Long, rodlike coiled-coils form the structural 
framework for many elongated proteins. Examples inelude a-keratin, 
which forms the intracellular fibers that reinforce the outer layer of the 
skin, and myosin, the motor protein responsible for muscle contraction 
(discussed in Chapter 17). 

p Sheets Form Rigid Structures at the Core of Many 

A p sheet is made when hydrogen bonds form between segments of a 
polypeptide chain that lie side by side (see Figure 4-13D). When the neigh- 
boring segments run in the same orientation (say, from the N-terminus to 
the C-terminus), the structure is a parallel ¡5 sheet; when they run in oppo- 
site directions, the structure is an antiparallel(i sheet (Figure 4-17). Both 
types of p sheet produce a very rigid, pleated structure, and they form the 
core of many proteins. Even the small bacterial protein HPr (see Figure 
4-12) contains several p sheets. 

P sheets have remarkable properties. They give silk fibers their extraor- 
dinary tensile strength. They also permit the formation of amyloid 
fibers —insoluble protein aggregates that inelude those associated with 
neurodegenerative disorders, such as Alzheimer's disease and prion dis- 
eases (see Figure 4-8). These structures, formed from abnormally folded 
proteins, are stabilized by p sheets that stack together tightly, with their 
amino acid side chains interdigitated like the teeth of a zipper (Figure 
4-18). Although we tend to associate amyloid fibers with disease, many 
organisms take advantage of these stable structures to perform novel 
tasks. Infectious bacteria, for example, can use amyloid fibers to help 
form the biofilms that allow them to colonize host tissues. Other types of 
filamentous bacteria use amyloid fibers to extend filaments into the air, 
enabling the bacteria to disperse their spores far and wide. 

Proteins Have Several Levels of Organizaron 

A protein's structure does not end with a hélices and p sheets; there are 
additional levels of organization. These levels are not independent but 
are built one upon the next to establish the three-dimensional structure 
of the entire protein. A protein's structure begins with its amino acid 
sequence, which is thus considered its primary structure. The next 
level of organization ineludes the a hélices and p sheets that form within 

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The Shape and Structure of Proteins 


Figure 4-16 Intertwined a hélices can 
form a stiff coiled-coil. In (A), a single a 
helix ¡s shown, with successive amino acid 
side chains labeled in a sevenfold repeating 
sequence "abcdefg." Amino acids "a" and 
"d" in such a sequence lie cióse together 
on the cylinder surface, forming a stripe 
(shaded in greer ) that winds slowly around 
the a helix. Proteins that form coiled-coils 
typically have nonpolar amino acids at 
positions "a" and "d." Consequently, as 
shown in (B), the two a hélices can wrap 
around each other, with the nonpolar side 
chains of one a helix interacting with the 
nonpolar side chains of the other, while the 
more hydrophilic amino acid side chains 
(shaded in red) are left exposed to the 
aqueous environment. (C) A portion of 
the atomic structure of a coiled-coil made 
of two a hélices, as determined by X-ray 
crystallography. In this structure, atoms that 
form the backbone of the hélices are shown 
in red; the interacting, nonpolar side chains 
are green, and the remaining side chains 
are gray. Coiled-coils can also form from 
three a hélices (Movie 4.3). 

certain segments of the polypeptide chain; these folds are elements of 
the protein's secondaiy structure. The full, three-dimensional confor- 
mation formed by an entire polypeptide chain—including the a hélices, 
P sheets, random coils, and any other loops and folds that form 
between the N- and C-termini—is sometimes referred to as the tertiary 
structure. Finally, if the protein molecule is formed as a complex of more 
than one polypeptide chain, then the complete structure is designated its 
quaternary structure. 

Studies of the conformation, function, and evolution of proteins have 
also revealed the importance of a level of organization distinct from 
the four just described. This organizational unit is the protein domain, 
which is defined as any segment of a polypeptide chain that can fold 
independently into a compact, stable structure. A protein domain usually 
contains between 40 and 350 amino acids—folded into a hélices and p 
sheets and other elements of secondary structure—and it is the modu¬ 
lar unit from which many larger proteins are constructed (Figure 4-19). 
The different domains of a protein are often associated with different 
functions. For example, the bacterial catabolite activator protein (CAP), 
illustrated in Figure 4-19, has two domains: the small domain binds to 
DNA, while the large domain binds cyclic AMP, a small intracellular sig- 
naling molecule. When the large domain binds cyclic AMP, it causes a 
conformational change in the protein that enables the small domain to 
bind to a speciflc DNA sequence and thereby promote the expression of 
an adjacent gene. To provide a sense of the many different domain struc- 
tures observed in proteins, ribbon models of three different domains are 
shown in Figure 4-20. 

Figure 4-17 P sheets come in two varieties. (A) Antiparallel p sheet 
(see also Figure 4-13D). (B) Parallel P sheet. Both of these structures 
are common in proteins. By convention, the arrows pointtoward the 
C-terminus of the polypeptide chain (Movie 4.4). 

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Protein Structure and Function 

Figure 4-18 Stacking of sheets allows 
some misfolded proteins to aggregate 
into amyloid fibers. (A) Electron 
micrograph shows an amyloid flberformed 
from a segment of a yeast prlon protein. 

(B) Schematlc representatlon shows the 
stacking of P sheets that stabillze an 
individual amyloid fiber. (A, courtesy of 
David Eisenberg.) 

Many Proteins Also Contain Unstructured Regions 

Small protein molecules, such as the oxygen-carrying muscle protein 
myoglobin, contain only a single domain (see Figure 4-11). Larger pro¬ 
teins can contain as many as several dozen domains, which are usually 
connected by relatively unstructured lengths of polypeptide chain. Such 
regions of polypeptide chain lacking any definite structure, which contin- 
ually bend and flex due to thermal buffeting, are abundant in cells. These 
intrinsically disordered sequences are often found as short stretches 
linking domains in otherwise highly ordered proteins. Other proteins, 
however, are almost entirely without secondary structure and exist as 
unfolded polypeptide chains in the cytosol. 

Intrinsically disordered sequences remained undetected for many years. 
Their lack of folded structure makes them prime targets for the prote- 
olytic enzymes that are released when cells are fractionated to isolate 
their molecular components (see Panel 4-3, pp. 164-165). Unstructured 
sequences also fail to form protein ciystals and for this reason escape 
the attention of X-ray crystallographers (see How We Know, pp. 162- 
163). Indeed, the ubiquity of disordered sequences became appreciated 
only after bioinformatics methods were developed that could recog- 
nize them from their amino acid sequences. Present estimates suggest 
that a third of all eukaryotic proteins have long unstructured regions in 
their polypeptide chain (greater than 30 amino acids in length), while a 
substantial number of eukaryotic proteins are mostly disordered under 
normal conditions. 

Unstructured sequences have a variety of important functions in cells. 
Being able to flex and bend, they can wrap around one or more target 
proteins like a scarf, binding with both high specificity and low affinity 
(Figure 4-21). By forming flexible tethers between the compact domains 
in a protein, they provide flexibility while increasing the frequency of 
encounters between the domains (Figure 4-21). They can help scaffold 
proteins bring together proteins in an intracellular signaling pathway, 
facilitating interactions (Figure 4-21). They also give proteins like elastin 

Figure 4-19 Many proteins are composed 
of sepárate functional domains. Elements 
of secondary structure such as a hélices 
and P sheets pack together into stable, 
¡ndependently folding, globular elements 
called protein domains. A typlcal protein 
molecule ¡s built from one or more domains, 
llnked by a reglón of polypeptide chain 
that ¡s often relatively unstructured. The 
ribbon diagram on the right represents the 
bacterial transcription regulator protein 
CAP, with one large domain (outlined in 
blue) and one small domain (outlined in 



LibertadDigital \ 2015 

The Shape and Structure of Proteins 


Figure 4-20 Ribbon models show 
three different protein domains. 

(A) Cytochrome bs62 ¡s a single-domain 
protein involved in electrón transfer in 
E coli. It is composed almost entirely of 
a hélices. (B) The NAD-binding domain 
of the enzyme lactic dehydrogenase ¡s 
composed of a mixture of a hélices and 
(3 sheets. (C) An ¡mmunoglobulin domain 
of an antibody molecule is composed of 
a sandwich of two antiparallel P sheets. In 
these examples, the a hélices are shown in 
green, while strands organized as P sheets 
are denoted by red arrows. The protruding 
loop regions ( yellow ) are often unstructured 
and can provide binding sites for other 
molecules. (Redrawn from origináis courtesy 
of Jane Richardson.) 

the ability to form rubberlike fibers, allowing our tendons and skin to 
recoil after being stretched. In addition to providing structural flexibil- 
ity, unstructured sequences are also ideal substrates for the addition of 
Chemical groups that control the way many proteins behave—a topic we 
discuss at length later in the chapter. 

Few of the Many Possible Polypeptide Chains 
Will Be Useful 

In theory, a vast number of different polypeptide chains could be made 
from 20 different amino acids. Because each amino acid is chemically 
distinct and could, in principie, occur at any position, a polypeptide chain 
four amino acids long has 20 x 20 x 20 x 20 = 160,000 different pos¬ 
sible sequences. In other words, for a polypeptide that is n amino acids 
long, 20 71 different chains are possible. For a typical protein length of 300 
amino acids, more than 20 300 (that's 10 390 ) different polypeptide chains 
could theoretically be made. 

Of the unimaginably large collection of potential polypeptide sequences, 
only a miniscule fraction is actually present in cells. That’s because many 
biological functions depend on proteins with stable, well-defined three- 
dimensional conformations. This requirement restricts the list of possible 
polypeptide sequences. Another constraint is that functional proteins 

Figure 4-21 Unstructured regions of a 
polypeptide chain in proteins can peform 
many functions. A few of these functions 
are ¡llustrated here. 

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CHAPTER 4 Protein Structure and Function 

must be "well-behaved" and not engage in unwanted associations with 
other proteins in the cell—forming insoluble protein aggregates, for 
example. Many potential proteins would therefore have been eliminated 
by natural selection through the long trial-and-error process that under- 
lies evolution (discussed in Chapter 9). 

Thanks to this rigorous process of selection, the amino acid sequences of 
many present-day proteins have evolved to guarantee that the polypep- 
tide will adopt a stable conformation—one that bestows upon the protein 
the exact Chemical properties that will enable it to perform a particu¬ 
lar function. Such proteins are so precisely built that a change in even 
a few atoms in one amino acid can sometimes disrupt the structure of 
a protein and thereby elimínate its function. In fact, the structures of 
many proteins—and their constituent domains—are so stable and effec- 
tive that they have been conserved throughout evolution among many 
diverse organisms. The three-dimensional structures of the DNA-binding 
domains from the yeast a2 protein and the Drosophila Engrailed protein, 
for example, are almost completely superimposable, even though these 
organisms are separated by more than a billion years of evolution. Other 
proteins, however, have changed their structure and function over evolu- 
tionary time, as we now discuss. 

Proteins Can Be Classified into Families 

Once a protein had evolved a stable conformation with useful proper¬ 
ties, its structure could be modified over time to enable it to perform 
new functions. We know that this occurred quite often during evolution, 
because many present-day proteins can be grouped into protein fami¬ 
lies, in which each family member has an amino acid sequence and a 
three-dimensional conformation that closely resemble those of the other 
family members. 

Consider, for example, the scrine proteases, a family of protein-cleaving 
(proteolytic) enzymes that ineludes the digestive enzymes chymotrypsin, 
tiypsin, and elastase, as well as several proteases involved in blood clot- 
ting. When any two of these enzymes are compared, portions of their 
amino acid sequences are found to be nearly the same. The similarity 
of their three-dimensional conformations is even more striking: most of 
the detailed twists and turns in their polypeptide chains, which are sev¬ 
eral hundred amino acids long, are virtually identical (Figure 4-22). The 
various serine proteases nevertheless have distinct enzymatic activities, 
each cleaving different proteins or the peptide bonds between different 
types of amino acids. 

Figure 4-22 Serine proteases constitute a 
family of proteolytic enzymes. Backbone 
models of two serine proteases, elastase 
and chymotrypsin, are ¡llustrated. Although 
only those amino acid sequences in the 
polypeptide chain shaded in green are 
the same in the two proteins, the two 
conformations are very similar nearly 
everywhere. Nonetheless, the two proteases 
prefer different substrates. The active site 
of each enzyme—where its substrates are 
bound and cleaved—is circled in red. 

Serine proteases derive their ñame 
from the amino acid serine, which directly 
participates in the cleavage reaction. The 
two black dots on the right s/de of the 
chymotrypsin molecule markthe two ends 
created where the enzyme has cleaved its 
own backbone. 


Random mutations only very rarely 
result in changes in a protein that 
improve its usefulness for the cell, 
yet useful mutations are selected in 
evolution. Because these changes 
are so rare, for each useful mutation 
there are innumerable mutations 
that lead to either no improvement 
or inactive proteins. Why, then, do 
cells not contain millions of proteins 
that are of no use? 

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The Shape and Structure of Proteins 


Large Protein Molecules Often Contain More Than 
One Polypeptide Chain 

The same type of weak noncovalent bonds that enable a polypeptide 
chain to fold into a specific conformation also allow proteins to bind 
to each other to produce larger structures in the cell. Any región on a 
protein's surface that interacts with another molecule through sets of 
noncovalent bonds is termed a bindingsite. A protein can contain binding 
sites for a variety of molecules, large and small. If a binding site recog- 
nizes the surface of a second protein, the tight binding of two folded 
polypeptide chains at this site will create a larger protein, whose quater- 
nary structure has a precisely defined geometry. Each polypeptide chain 
in such a protein is called a subunit, and each subunit may contain more 
than one domain. 

In the simplest case, two identical, folded polypeptide chains form a sym- 
metrical complex of two protein subunits (called a dimer) that is held 
together by interactions between two identical binding sites. The CAP 
protein in bacterial cells is a dimer (Figure 4-23A) formed frorn two iden¬ 
tical copies of the protein subunit shown previously in Figure 4-19. Many 
other symmetrical protein complexes, formed from múltiple copies of 
the same polypeptide chain, are commonly found in cells. The enzyme 
neuraminidase, for example, consists of a ring of four identical protein 
subunits (Figure 4-23B). 

Figure 4-23 Many protein molecules contain múltiple copies of the same protein subunit. (A) A symmetrical dimer. The CAP 
protein is a complex of two identical polypeptide chains (see also Figure 4-19). (B) A symmetrical homotetramer. The enzyme 
neuraminidase exists as a ring of four identical polypeptide chains. For both (A) and (B), a small schematic below the structure 
emphasizes how the repeated use of the same binding ¡nteraction forms the structure. In (A), the use of the same binding site on each 
monomer (represented by brown and green ovals) causes the formation of a symmetrical dimer. In (B), a pair of nonidentical binding 
sites (represented by orange áreles and blue sguares) causes the formation of a symmetrical tetramer. 

LibertadDigital \ 2015 


CHAPTER 4 Protein Structure and Function 

Figure 4-24 Some proteins are formed as 
a symmetrical assembly of two different 
subunits. Hemoglobin, an oxygen-carrying 
protein abundant ¡n red blood cells, 
contains two copies of a-globin (green) and 
two copies of P-globin (b/ue). Each of these 
four polypeptide chains contains a heme 
molecule (red), where oxygen (O2) ¡s bound. 
Thus, each molecule of hemoglobin ¡n the 
blood carries four molecules of oxygen. 









Other proteins contain two or more different polypeptide chains. 
Hemoglobin, the protein that carries oxygen in red blood cells, is a partic- 
ularly well-studied example. The protein contains two identical a-globin 
subunits and two identical p-globin subunits, symmetrically arranged 
(Figure 4-24). Many proteins contain múltiple subunits, and they can be 
veiy large (Movie 4.5). 

Proteins Can Assemble into Filaments, Sheets, or Spheres 

Proteins can form even larger assemblies than those discussed so far. 
Most simply, a chain of identical protein molecules can be formed if 
the binding site on one protein molecule is complementary to another 
región on the surface of another protein molecule of the same type. 
Because each protein molecule is bound to its neighbor in an identical 
way (see Figure 4-14), the molecules will often be arranged in a helix 
that can be extended indefinitely in either direction (Figure 4-25). This 
type of arrangement can produce an extended protein filament. An actin 
fllament, for example, is a long, helical structure formed from many mol¬ 
ecules of the protein actin (Figure 4-26). Actin is extremely abundant 
in eukaryotic cells, where it forms one of the major filament Systems of 
the cytoskeleton (discussed in Chapter 17). Other sets of identical pro¬ 
teins associate to form tubes, as in the microtubules of the cytoskeleton 
(Figure 4-27), or cagelike spherical shells, as in the protein coats of virus 
particles (Figure 4-28). 

Many large structures, such as virases and ribosomes, are built from a 
mixture of one or more types of protein plus RNA or DNA molecules. 
These structures can be isolated in puré form and dissociated into their 
constituent macromolecules. It is often possible to mix the isolated com- 
ponents back together and watch them reassemble spontaneously into 
the original structure. This demonstrates that all the information needed 
for assembly of the complicated structure is contained in the macro¬ 
molecules themselves. Experiments of this type show that much of the 

Figure 4-25 Identical protein subunits can assemble into complex 
structures. (A) A protein with just one binding site can form a dimer 
with another identical protein. (B) Identical proteins with two different 
binding sites will often form a long, helical filament. (C) If the two 
binding sites are disposed appropriately in relation to each other, the 
protein subunits will form a closed ring instead of a helix (see also 
Figure 4—23B). 

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The Shape and Structure of Proteins 


Figure 4-26 An actin filament is 
composed of ¡dentical protein subunits. 

The helical array of actin molecules ¡n 
a filament often contains thousands of 
molecules and extends for micrometers ¡n 
the cell. 

structure of a cell is self-organizing: if the required proteins are produced 
in the right amounts, the appropriate structures will form automatically. 

Some Types of Proteins Have Elongated Fibrous Shapes 

Most of the proteins we have discussed so far are globular proteins, in 
which the polypeptide chain folds up into a compact shape like a hall with 
an irregular surface. Enzymes, for example, tend to be globular proteins: 
even though many are large and complicated, with múltiple subunits, 
most have a quaternary structure with an overall rounded shape (see 
Figure 4-11). In contrast, other proteins have roles in the cell that require 
them to span a large distance. These proteins generally have a rela- 
tively simple, elongated three-dimensional structure and are commonly 
referred to as fibrous proteins. 

One large class of intracellular fibrous proteins resembles a-keratin, 
which we met earlier when we introduced the a-helix. Keratin filaments 
are extremely stable: long-lived structures such as hair, horns, and nails 
are composed mainly of this protein. An a-keratin molecule is a dimer 
of two identical subunits, with the long a hélices of each subunit form- 
ing a coiled-coil (see Figure 4-16). These coiled-coil regions are capped 
at either end by globular domains containing binding sites that allow 
them to assemble into ropelike intermedíate filaments —a component 
of the cytoskeleton that gives cells mechanical strength (discussed in 
Chapter 17). 

Fibrous proteins are especially abundant outside the cell, where they 
form the gel-like extracellular matrix that helps bind cells together to form 
tissues. These proteins are secreted by the cells into their surroundings, 
where they often assemble into sheets or long fibrils. Collagen is the most 
abundant of these fibrous extracellular proteins in animal tissues. A colla- 
gen molecule consists of three long polypeptide chains, each containing 
the nonpolar amino acid glycine at every third position. This regular struc¬ 
ture allows the chains to wind around one another to generate a long, 
regular, triple helix with glycine at its core (Figure 4-29A). Many such 

Figure 4-27 A single type of protein subunit can pack together to form a 
filament, a hollow tube, or a spherical shell. Actin subunits, for example, form 
actin filaments (see Figure 4-26), whereas tubulin subunits form hollow microtubules, 
and some virus proteins form a spherical shell (capsid) that endoses the viral genome 
(see Figure 4-28). 

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CHAPTER 4 Protein Structure and Function 

20 nm 

Figure 4-28 Many viral capsids are more 
or less spherical protein assemblies. 

They are formed from many copies of a 
small set of protein subunits. The nucleic 
acid of the virus (DNA or RNA) ¡s packaged 
inside. The structure of the simian virus 
SV40, shown here, was determined by X-ray 
crystallography and is known in atomic 
detail. (Courtesy of Robert Grant, Stephan 
Crainic, and James M. Hogle.) 

collagen molecules bind to one another side-by-side and end-to-end to 
create long overlapping arrays called collagen flbríls, which are extremely 
strong and help hold tissues together, as described in Chapter 20. 

In complete contrast to collagen is another fibrous protein in the extracel- 
lular matrix, elastin. Elastin molecules are formed from relatively loose 
and unstructured polypeptide chains that are covalently cross-linked into 
a rubberlike elastic meshwork. The resulting elasticfibers enable skin and 
other tissues, such as arteries and lungs, to stretch and recoil without 
tearing. As illustrated in Figure 4-29B, the elasticity is due to the ability 
of the individual protein molecules to uncoil reversibly whenever they 
are stretched. 

Extracellular Proteins Are Often Stabilized by Covalent 

Many protein molecules are either attached to the outside of a cell's 
plasma membrane or secreted as part of the extracellular matrix, which 
exposes them to extracellular conditions. To help maintain their struc- 
tures, the polypeptide chains in such proteins are often stabilized by 
covalent cross-linkages. These linkages can either tie together two amino 
acids in the same polypeptide chain or join together many polypeptide 
chains in a large protein complex—as for the collagen fibrils and elastic 
fibers just described. 

The most common covalent cross-links in proteins are sulfur-sulfur 
bonds. These disulfide bonds (also called S-S bonds) are formed before 
a protein is secreted by an enzyme in endoplasmic reticulum that links 
together two -SH groups from cysteine side chains that are adjacent in the 
folded protein (Figure 4-30). Disulfide bonds do not change a protein's 
conformation, but instead act as a sort of "atomic staple" to reinforce 
the protein's most favored conformation. For example, lysozyme—an 


elastic fiber 

Figure 4-29 Collagen and elastin are abundant extracellular fibrous proteins. (A) A collagen molecule is a triple helix formed 
by three extended protein chains that wrap around one another. Many rodlike collagen molecules are cross-linked together in the 
extracellular space to form collagen fibrils (top), which have the tensile strength of Steel. The striping on the collagen fibril is caused by 
the regular repeating arrangement of the collagen molecules within the fibril. (B) Elastin molecules are cross-linked together by covalent 
bonds (red) to form rubberlike, elastic fibers. Each elastin polypeptide chain uncoils into a more extended conformation when the fiber 
is stretched, and recoils spontaneously as soon as the stretching forcé is relaxed. 

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How Proteins Work 


polypeptide 2 

Figure 4-30 Disulfide bonds help stabilize 
a favored protein conformation. This 
diagram ¡llustrates how covalent disulfide 
bonds form between adjacent cysteine side 
chains by the oxidation oftheir-SH groups. 
As indicated, these cross-links can join 
eithertwo parts ofthe same polypeptide 
chain ortwo different polypeptide chains. 
Because the energy required to break one 
covalent bond is much largerthan the 
energy required to break even a whole set 
of noncovalent bonds (see Table 2-1, p. 48), 
a disulfide bond can have a major stabilizing 
effect on a protein's folded structure 
(Movie 4.6). 

enzyme in tears, saliva, and other secretions that can disrupt bacterial 
cell walls—retains its antibacterial activity for a long time because it is 
stabilized by such disulfide cross-links. 

Disulfide bonds generally do not form in the cell cytosol, where a high 
concentration of reducing agents converts such bonds back to cysteine 
-SH groups. Apparently, proteins do not require this type of structural 
reinforcement in the relatively mild conditions in the cytosol. 


As we have just seen, proteins are made frorn an enormous variety of 
amino acid sequences and can fold into a unique shape. The surface 
topography of a protein's side chains endows each protein with a unique 
function, based on its Chemical properties. The unión of structure, chem- 
istry, and function gives proteins the extraordinaiy ability to orchestrate 
the large number of dynamic processes that occur in cells. 

Thus, for proteins, form and function are inextricably linked. But the 
fundamental question remains: How do proteins actually work? In this 
section, we will see that the activity of proteins depends on their ability 
to bind specifically to other molecules, allowing them to act as catalysts, 
structural supports, tiny motors, and so on. The examples we review here 
by no means exhaust the vast functional repertoire of proteins. However, 
the specialized functions of the proteins you will encounter elsewhere in 
this book are based on the same principies. 

All Proteins Bind to Other Molecules 

The biological properties of a protein molecule depend on its physical 
interaction with other molecules. Antibodies attach to viruses or bacteria 
as part of the body's defenses; the enzyme hexokinase binds glucose and 
ATP to catalyze a reaction between them; actin molecules bind to one 
another to assemble into long filaments; and so on. Indeed, all proteins 
stick, or bind, to other molecules in a specific manner. In some cases, this 
binding is veiy tight; in others, it is weak and short-lived. As we saw in 
Chapter 3, the affinity of an enzyme for its substrate is reflected in its Km: 
the lower the Km, the tighter the binding. 

Regardless of its strength, the binding of a protein to other biological 
molecules always shows great specificity : each protein molecule can bind 
to just one or a few molecules out of the many thousands of different 


Hair ¡s composed largely of fibers 
of the protein keratin. Individual 
keratin fibers are covalently cross- 
linked to one another by many 
disulfide (S-S) bonds. If curly hair is 
treated with mild reducing agents 
that break a few of the cross-links, 
pulled straight, and then oxidized 
again, it remains straight. Draw a 
diagram that ¡llustrates the three 
different stages of this Chemical and 
mechanical process at the level of 
the keratin filaments, focusing on 
the disulfide bonds. What do you 
think would happen if hair were 
treated with strong reducing agents 
that break all the disulfide bonds? 

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CHAPTER 4 Protein Structure and Function 


noncovalent bonds 



Figure 4-31 The binding of a protein to 
another molecule is highly selective. Many 
weak interactions are needed to enable a 
protein to bind tightly to a second molecule 
(a ligand). The ligand musttherefore fit 
precisely into the protein's binding site, like 
a hand into a glove, so that a large number 
of noncovalent interactions can be formed 
between the protein and the ligand. 

(A) Schematic drawing shows the binding of 
a hypothetical protein and ligand; (B) space- 
filling model. 

molecules it encounters. Any substance that is bound by a protein— 
whether it is an ion, a small organic molecule, or a macromolecule—is 
referred to as a ligand for that protein (from the Latín ligare, "to bind”). 
The ability of a protein to bind selectively and with high afflnity to a ligand 
is due to the formation of a set of weak, noncovalent interactions—hydro- 
gen bonds, electrostatic attractions, and van der Waals attractions—plus 
favorable hydrophobic forces (see Panel 2-7, pp. 78-79). Each individ¬ 
ual noncovalent interaction is weak, so that effective binding requires 
many such bonds to be formed simultaneously. This is possible only if 
the surface contours of the ligand molecule fit very closely to the protein, 
matching it like a hand in a glove (Figure 4-31). 

When molecules have poorly matching surfaces, few noncovalent inter¬ 
actions occur, and the two molecules dissociate as rapidly as they come 
together. This is what prevenís incorrect and unwanted associations 
from forming between mismatched molecules. At the other extreme, 
when many noncovalent interactions are formed, the association can 
persist for a very long time. Strong binding between molecules occurs in 
cells whenever a biological function requires that the molecules remain 
tightly associated for a long time—for example, when a group of macro- 
molecules come together to form a functional subcellular structure such 
as a ribosome. 

The región of a protein that associates with a ligand, known as its bind¬ 
ing site, usually consists of a cavity in the protein surface formed by a 
particular arrangement of amino acid side chains. These side chains can 
belong to amino acids that are widely separated on the linear polypep- 
tide chain, but are brought together when the protein folds (Figure 4-32). 
Other regions on the surface often provide binding sites for different lig- 
ands that regúlate the protein's activity, as we discuss later. Yet other 
parts of the protein may be required to attract or attach the protein to a 
particular location in the cell—for example, the hydrophobic a helix of a 

am¡no acid 
side chains 

unfolded protein 

(A) folded protein 

Figure 4-32 Binding sites allow proteins to interact with specific ligands. (A) The folding of the polypeptide chain typically creates a 
crevice or cavity on the folded protein's surface, where specific amino acid side chains are brought together in such a way that they can 
form a set of noncovalent bonds only with certain ligands. (B) Close-up view of an actual binding site showing the hydrogen bonds and an 
electrostatic interaction formed between a protein and its ligand (in this example, the bound ligand is cyclic AMP, shown in darle brown). 

How Proteins Work 


membrane-spanning protein allows it to be inserted into the lipid bilayer 
of a cell membrane (discussed in Chapter 11). 

Although the atoms buried in the interior of a protein have no direct 
contact with the ligand, they provide an essential scaffold that gives the 
surface its contours and Chemical properties. Even tiny changes to the 
amino acids in the interior of a protein can change the protein's three- 
dimensional shape and destroy its function. 

There Are Billions of Different Antibodies, Each with a 
Different Binding Site 

All proteins must bind to particular ligands to carry out their various func- 
tions. For antibodies, the universe of possible ligands is limitless. Each of 
us has the capacity to produce a huge variety of antibodies, among which 
there will be one that is capable of recognizing and binding tightly to 
almost any molecule imaginable. 

Antibodies are immunoglobulin proteins produced by the immune Sys¬ 
tem in response to foreign molecules, especially those on the surface 
of an invading microorganism. Each antibody binds to a particular tar- 
get molecule extremely tightly, either inactivating the target directly or 
marking it for destruction. An antibody recognizes its target molecule— 
called an antigen—with remarkable specificity, and, because there are 
potentially billions of different antigens that a person might encounter, 
we have to be able to produce billions of different antibodies. 

Antibodies are Y-shaped molecules with two identical antigen-binding 
sites, each of which is complementary to a small portion of the surface 
of the antigen molecule. A detailed examination of the antigen-binding 
sites of antibodies reveáis that they are formed from several loops of 
polypeptide chain that protrude from the ends of a pair of closely juxta- 
posed protein domains (Figure 4-33). The amino acid sequence in these 

Figure 4-33 An antibody ¡s Y-shaped 
and has two identical antigen-binding 
sites, one on each arm of the Y. 

(A) Schematic drawing of a typical antibody 
molecule. The protein is composed of four 
polypeptide chains (two identical heavy 
chains and two identical and smaller light 
chains), held together by disulfide bonds 
(red). Each chain is made up of several 
similar domains, here shaded either blue 
or gray. The antigen-binding site is formed 
where a heavy-chain variable domain (Vh) 
and a light-chain variable domain (Vl) come 
cióse together. These are the domains that 
differ most in their amino acid sequence 

in different antibodies—henee their ñame. 

(B) Ribbon drawing of a single light chain 
showing that the most variable parís of the 
polypeptide chain ( orange ) extend as loops 
at one end of the variable domain (Vl) to 
form half of one antigen-binding site of the 
antibody molecule shown in (A). Note that 
both the constant and variable domains are 
composed of a sandwich of two antiparallel 
P sheets (see also Figure 4-20C), connected 
by a disulfide bond (red). 

variable domain 
of light chain (V L ) 

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Protein Structure and Function 


Use drawings to explain how 
an enzyme (such as hexokinase, 
mentioned in the text) can 
distinguish its normal substrate (here 
D-glucose) from the optical isomer 
L-glucose, which is not a substrate. 
(Hint: remembering that a carbón 
atom forms four single bonds 
that are tetrahedrally arranged 
and that the optical isomers are 
mirror images of each other around 
such a bond, draw the substrate 
as a simple tetrahedron with four 
different corners and then draw its 
mirror image. Using this drawing, 
indícate why only one optical isomer 
might bind to a schematic active site 
of an enzyme.) 

loops can vary greatly without altering the basic structure of the anti- 
body. An enormous diversity of antigen-binding sites can be generated by 
changing only the length and amino acid sequence of the loops, which is 
how the wide variety of different antibodies is formed (Movie 4.7). 

With their unique combination of specificity and diversity, antibodies are 
not only indispensable for fighting off infections, they are also invaluable 
in the laboratory, where they can be used to identify, purify, and study 
other molecules (Panel 4-2, pp. 146-147). 

Enzymes Are Powerful and Highly Specific Catalysts 

For many proteins, binding to another molecule is their main function. 
An actin molecule, for example, need only associate with other actin 
molecules to form a filament. There are proteins, however, for which lig- 
and binding is simply a necessary first step in their function. This is the 
case for the large and very important class of proteins called enzymes. 
These remarkable molecules are responsible for nearly all of the Chemi¬ 
cal transformations that occur in cells. Enzymes bind to one or more 
ligands, called substrates, and convert them into chemically modified 
producís, doing this over and over again with amazing rapidity. As we 
saw in Chapter 3, they speed up reactions, often by a factor of a mil- 
lion or more, without themselves being changed—that is, enzymes act as 
catalysts that permit cells to make or break covalent bonds at will. This 
catalysis of organized sets of Chemical reactions by enzymes creates and 
maintains the cell, making life possible. 

Enzymes can be grouped into functional classes based on the Chemi¬ 
cal reactions they catalyze (Table 4-1). Each type of enzyme is highly 
specific, catalyzing only a single type of reaction. Thus, hexokinase adds 
a phosphate group to D-glucose but not to its optical isomer L-glucose; 
the blood-clotting enzyme thrombin cuts one type of blood-clotting pro¬ 
tein between a particular arginine and its adjacent glycine and nowhere 


1 Enzyme Class 

1 Biochemical Function 


General term for enzymes that catalyze a hydrolytic cleavage reaction 


Breaks down nucleic acids by hydrolyzing bonds between nucleotides 


Breaks down proteins by hydrolyzing peptide bonds between amino acids 


Joins two molecules together; DNA ligase joins two DNA strands together end-to-end 


Catalyzes the rearrangement of bonds within a single molecule 


Catalyzes polymerization reactions such as the synthesis of DNA and RNA 


Catalyzes the addition of phosphate groups to molecules. Protein kinases are an important group of kinases 
that attach phosphate groups to proteins 


Catalyzes the hydrolytic removal of a phosphate group from a molecule 


General ñame for enzymes that catalyze reactions in which one molecule is oxidized while the other is 
reduced. Enzymes of this type are often called oxidases, reductases, or dehydrogenases 


Flydrolyzes ATP. Many proteins have an energy-harnessing ATPase activity as part of their function, including 
motor proteins such as myosin (discussed in Chapter 17) and membrane transport proteins such as the 
sodium pump (discussed in Chapter 12) 

Enzyme ñames typicall; 
which were discovered 

y end in "-ase," with the exception of some enzymes, such as pepsin, trypsin, thrombin, lysozyme, and so on, 
and named before the convention became generally accepted atthe end of the nineteenth century. The ñame 

of an enzyme usually ¡ndicates the nature of the reaction catalyzed. For example, citrate synthase catalyzes the synthesis of citrate by 
reaction between acetyl CoA and oxaloacetate. 

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How Proteins Work 


else. As discussed in detail in Chapter 3, enzymes often work in tándem, 
with the product of one enzyme becoming the substrate for the next. The 
result is an elabórate network oímetabolic pathways that provides the cell 
with energy and generates the many large and small molecules that the 
cell needs. 

Lysozyme lllustrates How an Enzyme Works 

To explain how enzymes catalyze Chemical reactions, we will use the 
example of lysozyme—an enzyme that acts as a natural antibiotic in egg 
white, saliva, tears, and other secretions. Lysozyme severs the polysac- 
charide chains that form the cell walls of bacteria. Because the bacterial 
cell is under pressure due to intracellular osmotic forces, cutting even a 
small number of polysaccharide chains causes the cell wall to rupture 
and the bacterium to burst, or lyse. Lysozyme is a relatively small and 
stable protein, which can be isolated easily in large quantities. For these 
reasons it has been intensively studied, and it was the first enzyme whose 
structure was worked out in atomic detail by X-ray crystallography. 

The reaction catalyzed by lysozyme is a hydrolysis: the enzyme adds a 
molecule of water to a single bond between two adjacent sugar groups in 
the polysaccharide chain, thereby causing the bond to break. The reaction 
is energetically favorable because the free energy of the severed polysac¬ 
charide chain is lower than the free energy of the intact chain. However, 
the puré polysaccharide can sit for years in water without being hydro- 
lyzed to any detectable degree. This is because there is an energy barrier 
to such reactions, called the activation energy (discussed in Chapter 3, 
pp. 91-93). For a colliding water molecule to break a bond linking two 
sugars, the polysaccharide molecule has to be distorted into a particular 
shape—the transition State—in which the atoms around the bond have 
an altered geometry and electrón distribution. To distort the polysaccha¬ 
ride in this way requires a large input of energy from random molecular 
collisions. In aqueous solution at room temperature, the energy of such 
collisions almost never exceeds the activation energy; therefore, hydroly¬ 
sis occurs extremely slowly, if at all. 

This is where the enzyme comes in. Like all enzymes, lysozyme has a 
binding site on its surface, termed an active site, that eradles the con- 
tours of its substrate molecule. Here, the catalysis of the Chemical reaction 
occurs. Because its substrate is a polymer, lysozyme’s active site is a long 
groove that holds six linked sugars in the polysaccharide chain at the 
same time. As soon as the enzyme-substrate complex forms, the enzyme 
cuts the polysaccharide by catalyzing the addition of a water molecule to 
one of its sugar-sugar bonds. The severed chain is then quickly released, 
freeing the enzyme for further eyeles of cleavage (Figure 4-34). 

The chemistiy that underlies the binding of lysozyme to its substrate is 
the same as that for antibody binding to its antigen: the formation of 


(A) S + E -► ES _► EP 


Figure 4-34 Lysozyme cleaves a 
polysaccharide chain. (A) Schematic view 
of the enzyme lysozyme (E), which catalyzes 
the cutting of a polysaccharide substrate 
molecule (S). The enzyme first binds to 
the polysaccharide to form an enzyme- 
substrate complex (ES), then it catalyzes the 
cleavage of a specific covalent bond 
in the backbone of the polysaccharide. 

The resulting enzyme-product complex 
(EP) rapidly dissociates, releasing the 
producís (P) and leaving the enzyme free 
to act on another substrate molecule. 

(B) A space-filling model of lysozyme bound 
to a short length of polysaccharide chain 
prior to cleavage. (B, courtesy of 
Richard J. Feldmann.) 

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How Proteins Work 



CHAPTER 4 Protein Structure and Function 


This substrate is an oligosaccharide of six sugars, The final producís are an oligosaccharide of four sugars 

labeled A through F. Only sugars D and E are shown in detail. (left) and a disaccharide (right), produced by hydrolysis. 

Figure 4-35 Enzymes bind to, and 
chemically alter, substrate molecules. 

In the active site of lysozyme, a covalent 
bond in a polysaccharide molecule is bent 
and then broken. The top row shows the 
free substrate and the free producís. The 
three lower panels depict sequential events 
at the enzyme active site, during which a 
sugar-sugar covalent bond ¡s broken. Note 
the change in the conformation of sugar D 
in the enzyme-substrate complex compared 
with the free substrate. This conformation 
favors the formation of the transition State 
shown in the middle panel, greatly lowerlng 
the activation energy required for the 
reactlon. The reaction, and the structure of 
lysozyme bound to its product, are shown in 
Movie 4.8 and Movie 4.9. (Based on 
D.J. Vocadlo et al., Nature 412:835-838, 

múltiple noncovalent bonds. However, lysozyme holds its polysaccharide 
substrate in such a way that one of the two sugars involved in the bond 
to be broken is distorted from its normal, most stable conformation. The 
bond to be broken is held cióse to two specific amino acids with acidic 
side chains—a glutamic acid and an aspartic acid—located within the 
active site of the enzyme. Conditions are thereby created in the microen- 
vironment of the lysozyme active site that greatly reduce the activation 
energy necessary for the hydrolysis to take place (Figure 4-35). The over- 
all Chemical reaction, from the initial binding of the polysaccharide on the 
surface of the enzyme to the final release of the severed chains, occurs 
many millions of times faster than it would in the absence of enzyme. 
Other enzymes use similar mechanisms to lower the activation energies 
and speed up the reactions they catalyze. In reactions involving two or 
more substrates, the active site also acts like a témplate or mold that 
brings the reactants together in the proper orientation for the reaction 
to occur (Figure 4-36A). As we saw for lysozyme, the active site of an 
enzyme contains precisely positioned Chemical groups that speed up the 
reaction by altering the distribution of electrons in the substrates (Figure 
4-36B). Binding to the enzyme also changes the shape of the substrate, 
bending bonds so as to drive the bound molecule toward a particular 
transition State (Figure 4-36C). Finally, like lysozyme, many enzymes 
particípate intimately in the reaction by briefly forming a covalent bond 
between the substrate and an amino acid side chain in the active site. 
Subsequent steps in the reaction restore the side chain to its original 
State, so the enzyme remains unchanged after the reaction and can go on 
to catalyze many more reactions. 

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How Proteins Work 


Figure 4-36 Enzymes can encourage 
a reaction in several ways. (A) Holding 
reacting substrates together ¡n a precise 
alignment. (B) Rearranging the distribution 
of charge ¡n a reaction intermedíate. 

(C) Altering bond angles in the substrate to 
increase the rate of a particular reaction. 

(A) enzyme binds to two 

(B) binding of substrate 
to enzyme rearranges 

(C) enzyme strains the 
bound substrate 
molecule, forcing it 
toward a transition 

substrate molecules and 
orients them precisely to 
encourage a reaction to 
occur between them 

electrons in the substrate, 
creating partial negative 

and positive charges 
that favor a reaction 

State to favor a reaction 

Many Drugs Inhibit Enzymes 

Many of the drugs we take to treat or prevent illness work by blocking the 
activity of a particular enzyme. Cholesterol-lowering statins inhibit HMG- 
CoA reducíase, an enzyme involved in the synthesis of cholesterol by 
the liver. Methotrexate kills some types of cáncer cells by shutting down 
dihydrofolate reducíase, an enzyme that produces a compound required 
for DNA synthesis during cell división. Because cáncer cells have lost 
important intracellular control systems, some of them are unusually sen- 
sitive to treatments that interrupt chromosome replication, making them 
susceptible to methotrexate. 

Pharmaceutical companies often develop drugs by first using automated 
methods to screen massive libraries of compounds to find Chemicals that 
are able to inhibit the activity of an enzyme of interest. They can then 
chemically modify the most promising compounds to make them even 
more effective, enhancing their binding affinity and specificity for the tar- 
get enzyme. As we discuss in Chapter 20, the anticancer drug Gleevec® 
was designed to speciflcally inhibit an enzyme whose aberrant behav- 
ior is required for the growth of a type of cáncer called chronic myeloid 
leukemia. The drug binds tightly in the substrate-binding pocket of the 
enzyme, blocking its activity (see Figure 20-56). 

Tightly Bound Small Molecules Add Extra Functions to 

Although the order of amino acids in proteins gives these macromol- 
ecules their shape and functional versatility, sometimes the amino acids 
by themselves are not enough for a protein to do its job. Just as we use 
tools to enhance and extend the capabilities of our hands, so proteins 
often employ small, nonprotein molecules to perform functions that 
would be difficult or impossible using amino acids alone. Thus, the pho- 
toreceptor protein rhodopsin, which is the light-sensitive protein made 
by the rod cells in the retina, detects light by means of a small molecule, 
retinal, which is attached to the protein by a covalent bond to a lysine side 
chain (Figure 4-37A). Retinal changes its shape when it absorbs a photon 
of light, and this change is amplified by rhodopsin to trigger a cascade of 
reactions that eventually leads to an electrical signal being carried to the 

Another example of a protein that contains a nonprotein portion essential 
for its function is hemoglobin (see Figure 4-24). A molecule of hemoglobin 
carries four noncovalently bound heme groups, ring-shaped molecules 
each with a single central iron atom (Figure 4-37B). Heme gives hemo¬ 
globin (and blood) its red color. By binding reversibly to dissolved oxygen 
gas through its iron atom, heme enables hemoglobin to pick up oxygen 
in the lungs and release it in tissues that need it. 

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CHAPTER 4 Protein Structure and Function 

Figure 4-37 Retinal and heme are 
required for the function of certain 
proteins. (A) The structure of retinal, the 
light-sensitive molecule covalently attached 
to the rhodopsin protein in our eyes. (B) The 
structure of a heme group, shown with the 
carbon-containing heme ring colored red 
and the ¡ron atom at its center in orange. 

A heme group is tightly, but noncovalently, 
bound to each of the four polypeptide 
chains in hemoglobin, the oxygen-carrying 
protein whose structure was shown in 
Figure 4-24. 

When these small molecules are attached to their protein, they become 
an integral part of the protein molecule itself. We discuss in Chapter 11 
how proteins can be anchored to cell membranes through covalently 
attached lipid molecules, and how proteins that are either secreted from 
the cell or bound to its surface can be modified by the covalent addition 
of sugars and oligosaccharides. 

Enzymes, too, make use of nonprotein molecules: they frequently have 
a small molecule or metal atom associated with their active site that 
assists with their catalytic function. Carboxypeptidase, an enzyme that 
cuts polypeptide chains, carries a tightly bound zinc ion in its active site. 
During the cleavage of a peptide bond by carboxypeptidase, the zinc ion 
forms a transient bond with one of the substrate atoms, thereby assist- 
ing the hydrolysis reaction. In other enzymes, a small organic molecule 
serves a similar purpose. Biotin, for example, is found in enzymes that 
transfer a carboxyl group (-COCr) from one molecule to another (see 
Figure 3-37). Biotin participates in these reactions by forming a transient 
covalent bond to the -COO“ group to be transferred, thereby forming an 
activated carrier (see Table 3-2, p. 112). This small molecule is better 
suited for this function than any of the amino acids used to make proteins. 
Because biotin cannot be synthesized by humans, it must be provided in 
the diet; thus biotin is classified as a vitamin. Other vitamins are similarly 
needed to make small molecules that are essential components of our 
proteins; vitamin A, for example, is needed in the diet to make retinal, the 
light-sensitive part of rhodopsin just discussed. 


So far, we have examined how proteins do their jobs: how binding to 
other proteins or small molecules allows them to perform their specific 
functions. But inside the cell, most proteins and enzymes do not work 
continuously, or at full speed. Instead, their activities are regulated in a 
coordinated fashion so the cell can maintain itself in an optimal State, 
producing only those molecules it requires to thrive under the current 
conditions. By coordinating when—and how vigorously—proteins func¬ 
tion, the cell ensures that it does not deplete its energy reserves by 
accumulating molecules it does not need or waste its stockpiles of criti- 
cal substrates. We now consider how cells control the activity of their 
enzymes and other proteins. 

The regulation of protein activity occurs at many levels. At one level, the 
cell Controls the amount of the protein it contains. It can do so by regu- 
lating the expression of the gene that encodes that protein (discussed in 
Chapter 8), and by regulating the rate at which the protein is degraded 

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How Proteins Are Controlled 


(discussed in Chapter 7). At another level, the cell Controls enzymatic 
activities by confining sets of enzymes to particular subcellular com- 
partments, often—but not always—enclosed by distinct membranes 
(discussed in Chapters 14 and 15). But the most rapid and general mech- 
anism used to adjust the activity of a protein occurs at the level of the 
protein itself. Although proteins can be switched on or oflf in various 
ways, as we see next, all of these mechanisms cause the protein to alter 
its shape, and therefore its function. 

The Catalytic Activities of Enzymes Are Often Regulated 
by Other Molecules 

A living cell contains thousands of different enzymes, many of which 
are operating at the same time in the same small volume of the cytosol. 
By their catalytic action, enzymes generate a complex web of metabolic 
pathways, each composed of chains of Chemical reactions in which the 
product of one enzyme becomes the substrate of the next. In this maze of 
pathways, there are many branch points where different enzymes com¬ 
pete for the same substrate. The System is so complex that elabórate 
Controls are required to regúlate when and how rapidly each reaction 

A common type of control occurs when a molecule other than a substrate 
specifically binds to an enzyme at a special regulatory site, altering the 
rate at which the enzyme converts its substrate to product. In feedback 
inhibition, for example, an enzyme acting early in a reaction pathway is 
inhibited by a late product of that pathway. Thus, whenever large quan- 
tities of the final product begin to accumulate, the product binds to an 
earlier enzyme and slows down its catalytic action, limiting further entry 
of substrates into that reaction pathway (Figure 4-38). Where pathways 
branch or intersect, there are usually múltiple points of control by dif¬ 
ferent final products, each of which works to regúlate its own synthesis 
(Figure 4-39). Feedback inhibition can work almost instantaneously and 
is rapidly reversed when product levels fall. 

Feedback inhibition is a negative regulation: it prevenís an enzyme from 
acting. Enzymes can also be subject to positive regulation, in which the 
enzyme's activity is stimulated by a regulatory molecule rather than being 
suppressed. Positive regulation occurs when a product in one branch 
of the metabolic maze stimulates the activity of an enzyme in another 

Allosteric Enzymes Have Two or More Binding Sites That 
Influence One Another 

One feature of feedback inhibition was initially puzzling to those who 
discovered it. Unlike what one expects to see for a competitive inhibitor 
(see Figure 3-29), the regulatory molecule often has a shape that is totally 
different from the shape of the enzyme's preferred substrate. Indeed, 
when this form of regulation was discovered in the 1960s, it was termed 
allostery (from the Greek alio, "other," and stere, "solid" or "shape"). As 
more was leamed about feedback inhibition, researchers realized that 
many enzymes must have at least two different binding sites on their sur- 
face: the active site that recognizes the substrates and one or more sites 
that recognize regulatory molecules. And that these sites must somehow 
"communicate" to allow the catalytic events at the active site to be influ- 
enced by the binding of the regulatory molecule at its sepárate site. 

The interaction between sites that are located in different regions on a 
protein molecule is now known to depend on conformational changes in 
the protein: binding of a ligand to one of the sites causes a shift in the pro- 
tein's structure from one folded shape to a slightly different folded shape, 

Figure 4-38 Feedback inhibition regulates 
the flow through biosynthetic pathways. 

B ¡s the first metabolite in a pathway that 
gives the end product Z. Z inhibits the first 
enzyme that is specific to its own synthesis 
and thereby limits its own concentraron ¡n 
the cell. This form of negative regulation is 
called feedback inhibition. 


Conslder the drawing in Figure 
4-38. What will happen if, instead of 
the indicated feedback, 

A. Feedback inhibition from Z 
affects the step B —> C only? 

B. Feedback inhibition from Z 
affects the step Y —► Z only? 

C. Z is a positive regulator of the 
step B —► X? 

D. Z is a positive regulator of the 
step B —» C? 

For each case, discuss how useful 
these regulatory schemes would be 
for a cell. 

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CHAPTER 4 Protein Structure and Function 

Figure 4-39 Feedback inhibition at 
múltiple points regulates connected 
metabolic pathways. The biosynthetic 
pathways for four different amino acids ¡n 
bacteria are shown, starting from the amino 
acid aspartate. The red lines indícate points 
at which producís feed back to inhibit 
enzymes and the blank boxes represent 
intermediates in each pathway. In this 
example, each amino acid Controls the first 
enzyme specific to its own synthesis, thereby 
limiting its own concentrations and avoiding 
a wasteful buildup of intermediates. Some 
ofthe producís also separately inhibit the 
initial set of reactions common to all the 
syntheses. Three different enzymes catalyze 
the initial reaction from aspartate to aspartyl 
phosphate, and each of these enzymes is 
inhibited by a different product. 


which alters the binding of a ligand to a second site. Many enzymes have 
two conformations that differ in activity, each stabilized by the binding 
of different ligands. During feedback inhibition, for example, the binding 
of an inhibitor at a regulatory site on the protein causes the protein to 
shift to a conformation in which its active site—located elsewhere in the 
protein—becomes less accommodating to the substrate molecule (Figure 

Many—if not most—protein molecules are allosteric: they can adopt two 
or more slightly different conformations, and their activity can be regu- 
lated by a shift from one to another. This is true not only for enzymes 
but also for many other proteins as well. The chemistry involved here 
is extremely simple in concept: because each protein conformation will 
have somewhat different contours on its surface, the protein's binding 
sites for ligands will be altered when the protein changes shape. Each lig¬ 
and will stabilize the conformation that it binds to most strongly, and at 
high enough concentrations a ligand will tend to "switch" the population 
of proteins to the conformation that it favors (Figure 4-41). 

Phosphorylation Can Control Protein Activity by 
Causing a Conformational Change 

Enzymes are regulated solely by the binding of small molecules. Another 
method that eukaryotic cells use with great frequency to regúlate protein 

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How Proteins Are Controlled 





activity involves attaching a phosphate group covalently to one or more 
of the protein's amino acid side chains. Because each phosphate group 
carries two negative charges, the enzyme-catalyzed addition of a phos¬ 
phate group can cause a major conformational change in a protein by, for 
example, attracting a cluster of positively charged amino acid side chains 
from somewhere else in the same protein. This conformational change 
can, in turn, affect the binding of ligands elsewhere on the protein sur- 
face, thereby altering the protein’s activity. Removal of the phosphate 
group by a second enzyme will retum the protein to its original confor- 
mation and restore its initial activity. 

This reversible protein phosphoiylation Controls the activity of many 
types of proteins in eukaiyotic cells; indeed, it is used so extensively that 
more than one-third of the 10,000 or so proteins in a typical mamma- 
lian cell are phosphorylated at any one time. The addition and removal 
of phosphate groups from specific proteins often occur in response to 
signáis that specify some change in a cell's State. For example, the com- 
plicated series of events that takes place as a eukaiyotic cell divides is 
timed largely in this way (discussed in Chapter 18). And many of the 
intracellular signaling pathways activated by extracellular signáis such 
as hormones depend on a network of protein phosphoiylation events 
(discussed in Chapter 16). 

Protein phosphorylation involves the enzyme-catalyzed transfer of the 
terminal phosphate group of ATP to the hydroxyl group on a serine, thre- 
onine, or tyrosine side chain of the protein. This reaction is catalyzed 

Figure 4-40 Feedback inhibition 
triggers a conformational change in an 
enzyme. The enzyme shown, aspartate 
transcarbamoylase from £. coli, was used 
¡n early studies of allosteric regulation. 

This large multisubunit enzyme (see Figure 
4-11) catalyzes an ¡mportant reaction that 
begins the synthesis of the pyrimidine 
ring of C, U, and T nucleoides (see Panel 
2-6, p. 76-77). One of the final producís of 
this pathway, cytosine triphosphate (CTP), 
binds to the enzyme to turn it off whenever 
CTP is plentiful. This diagram shows the 
conformational change that occurs when the 
enzyme is turned off by CTP binding to its 
four regulatory sites, which are distinct from 
the active site where the substrate binds. 
Note that the aspartate transcarbamoylase 
shown in Figure 4-11 is seen from the top. 
This figure depicts the enzyme as seen from 
the side. 

Figure 4-41 The equilibrium between two 
conformations of a protein is affected 
by the binding of a regulatory ligand. 

(A) Schematic diagram of a hypothetical, 
allosterically regulated enzyme for which a 
rise in the concentration of ADP molecules 
(red wedges) increases the rate at which 
the enzyme catalyzes the oxidation of sugar 
molecules (blue hexagons). (B) With no 
ADP present, only a small fraction of the 
enzyme molecules spontaneously adopt 
the active (closed) conformation; most 
are in the inactive (open) conformation. 

(C) Because ADP can bind to the protein 
only in its closed, active conformation, an 
increase in ADP concentration locks nearly 
all of the enzyme molecules in the active 
form. Such an enzyme could be used, for 
example, to sense when ADP is building up 
in the cell—which is usually a sign that ATP 
is decreasing. In this way, the increase in 
ADP would increase the oxidation of sugars 
to provide more energy for the synthesis 
of ATP from ADP—an example of positive 

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Protein Structure and Function 



(A) ® 

(B) <g) 


Figure 4-42 Protein phosphorylation is a 
very common mechanism for regulating 
protein activity. Many thousands of 
proteins in a typical eukaryotic cell are 
modified by the covalent addition of one 
or more phosphate groups. (A) The general 
reaction, shown here, entails transfer of a 
phosphate group from ATP to an amino 
acid side chain of the target protein by a 
protein kinase. Removal of the phosphate 
group is catalyzed by a second enzyme, a 
protein phosphatase. In this example, the 
phosphate ¡s added to a serine side chain; 
¡n other cases, the phosphate ¡s instead 
linked to the -OFI group of a threonine or 
tyrosine side chain. (B) Phosphorylation 
can either increase or decrease the 
protein's activity, depending on the site of 
phosphorylation and the structure of the 

by a protein kinase. The reverse reaction—removal of the phosphate 
group, or dephosphoiylation —is catalyzed by a protein phosphatase 
(Figure 4-42A). Phosphorylation can either stimulate protein activity or 
inhibit it, depending on the protein involved and the site of phosphoryla¬ 
tion (Figure 4-42B). Cells contain hundreds of different protein kinases, 
each responsible for phosphorylating a different protein or set of pro¬ 
teins. Cells also contain a smaller set of different protein phosphatases; 
some of these are highly specific and remove phosphate groups from only 
one or a few proteins, whereas others act on a broad range of proteins. 
The State of phosphorylation of a protein at any moment in time, and thus 
its activity, will depend on the relative activities of the protein kinases 
and phosphatases that act on it. 

For many proteins, a phosphate group is added to a particular side 
chain and then removed in a continuous cycle. Phosphorylation cycles 
of this kind allow proteins to switch rapidly from one State to another. 
The more rapidly the cycle is "turning," the faster the concentration of a 
phosphorylated protein can change in response to a sudden stimulus that 
increases its rate of phosphorylation. However, keeping the cycle turning 
costs energy, because one molecule of ATP is hydrolyzed with each turn 
of the cycle. 

Covalent Modifications Also Control the Location and 
Interaction of Proteins 

Phosphorylation can do more than control a protein's activity; it can 
create docking sites where other proteins can bind, thus promoting the 
assembly of proteins into larger complexes. For example, when extracel- 
lular signáis stimulate a class of cell-surface, transmembrane proteins 
called receptor tyrosine kinases, they cause the receptor proteins to phos- 
phorylate themselves on certain tyrosines. The phosphorylated tyrosines 
then serve as docking sites for the binding and activation of various 
intracellular signaling proteins, which pass along the message to the cell 
interior and change the behavior of the cell (see Figure 16-32). 
Phosphorylation is not the only form of covalent modification that can 
affect a protein's activity or location. More than 100 types of covalent 
modifications can occur in the cell, each playing its own role in regulat¬ 
ing protein function. Many proteins are modified by the addition of an 
acetyl group to a lysine side chain. And the addition of the fatty acid 
palmitate to a cysteine side chain drives a protein to associate with cell 
membranes. Attachment of ubiquitin, a 76-amino-acid polypeptide, can 
target a protein for degradation, as we discuss in Chapter 7. Each of these 
modifying groups is enzymatically added or removed depending on the 
needs of the cell. 

A large number of proteins are modified on more than one amino acid 
side chain. The p53 protein, which plays a central part in controling how 
a cell responds to DNA damage and other stresses, can be modified at 
20 sites (Figure 4-43). Because an enormous number of combinations of 
these 20 modifications is possible, the protein's behavior can in principie 
be altered in a huge number of ways. 

The set of covalent modifications that a protein contains at any moment 
constitutes an important form of regulation. The attachment or removal 
of these modifying groups Controls the behavior of a protein, changing its 
activity or stability, its binding partners, or its location inside the cell. In 
some cases, the modification alters the protein's conformation; in others, 
it serves as a docking site for other proteins to attach. This layer of con¬ 
trol enables the cell to make optimal use of its proteins, and it allows the 
cell to respond rapidly to changes in its environment. 

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"I r¿ 

p i phosphate JS&. acetyl w ubiquitin 

GTP-B¡nd¡ng Proteins Are Also Regulated by the Cyclic 
Gain and Loss of a Phosphate Group 

Eukaryotic cells have a second way to regúlate protein activity by phos¬ 
phate addition and removal. In this case, however, the phosphate is not 
enzymatically transferred from ATP to the protein. Instead, the phosphate 
is part of a guanine nucleotide—guanosine triphosphate (GTP)—that is 
bound tightly to various types of GTP-binding proteins. These proteins 
act as molecular switches: they are in their active conformation when 
GTP is bound, but they can hydrolyze this GTP to GDP, which releases a 
phosphate and flips the protein to an inactive conformation. As with pro¬ 
tein phosphorylation, this process is reversible: the active conformation 
is regained by dissociation of the GDP, followed by the binding of a fresh 
molecule of GTP (Figure 4-44). 

A large variety of such GTP-binding proteins function as molecular 
switches in cells. The dissociation of GDP and its replacement by GTP, 
which turns the switch on, is often stimulated in response to a signal 
received by the cell. The GTP-binding proteins in turn bind to other pro¬ 
teins to control their activities; their crucial role in intracellular signaling 
pathways is discussed in detail in Chapter 16. 

ATP Hydrolysis Allows Motor Proteins to Produce Directed 
Movements in Cells 

We have seen how conformational changes in proteins play a central 
part in enzyme regulation and cell signaling. But conformational changes 
also play another important role in the operation of the eukaryotic cell: 
they enable certain specialized proteins to drive directed movements of 
cells and their components. These motor proteins generate the forces 
responsible for muscle contraction and most other eukaryotic cell move¬ 
ments. They also power the intracellular movements of organdíes and 
macromolecules. For example, they help move chromosomes to opposite 
ends of the cell during mitosis (discussed in Chapter 18), and they move 
organdíes along cytoskeletal tracks (discussed in Chapter 17). 

How are shape changes in proteins used to generate such orderly move¬ 
ments? If, for example, a protein is required to walk along a cytoskeletal 
fiber, it can move by undergoing a series of conformational changes. 
However, with nothing to drive these changes in an orderly sequence, 
the shape changes will be perfectly reversible. Thus the protein can only 
wander randomly back and forth (Figure 4-45). 

GTP-binding protein 


How Proteins Are Controlled 155 

Figure 4-43 The modification of a 
protein at múltiple sites can control the 
protein's behavior. This diagram shows 
some of the covalent modifications that 
c control the activity and degradaron of the 
protein p53, an important gene regulatory 
protein that regulates a cell's response 
to damage (discussed in Chapter 18). 

Not all of these modifications will be 
present at the same time. Colors along 
the body of the protein represent distinct 
protein domains, ¡ncluding one that binds 
to DNA {greerí) and one that activates 
gene transcription (pink). All of the 
modifications shown are located within 
relatively unstructured regions of the 
polypeptide chain. 


Explain how phosphorylation and 
the binding of a nucleotide (such as 
ATP or GTP) can both be used to 
regúlate protein activity. What do 
you suppose are the advantages of 
either form of regulation? 

Figure 4-44 GTP-binding proteins 
function as molecular switches. A GTP- 
binding protein requires the presence 
of a tightly bound GTP molecule to be 
active (switch ON). The active protein can 
shut itself off by hydrolyzing its bound 
GTP to GDP and ¡norganic phosphate (P¡), 
which converts the protein to an inactive 
conformation (switch OFF). To reactívate 
the protein, the tightly bound GDP must 
dissociate, a slow step that can be greatly 
accelerated by specific signáis; once the 
GDP dissociates, a molecule of GTP quickly 
replaces it, returning the protein to its active 

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CHAPTER 4 Protein Structure and Function 


direction of 

Figure 4-45 Changes ¡n conformation can allow a protein to 
"walk" along a cytoskeletal filament. This protein's three different 
conformations allow ¡t to wander randomly back and forth while bound 
to a filament. Without an input of energy to drlve its movement ¡n a 
single direction, the protein wlll only shuffle almlessly, getting nowhere. 

To make the conformational changes unidirectional—and forcé the entire 
cycle of movement to proceed in one direction—it is enough to make 
any one of the steps irreversible. For most proteins that are able to move 
in a single direction for long distances, this irreversibility is achieved 
by coupling one of the conformational changes to the hydrolysis of an 
ATP molecule bound to the protein—which is why motor proteins are 
also ATPases. A great deal of free energy is released when ATP is hydro- 
lyzed, making it veiy unlikely that the protein will undergo a reverse 
shape change—as required for moving backward. (Such a reversal would 
require that the ATP hydrolysis be reversed, by adding a phosphate mol¬ 
ecule to ADP to form ATP.) As a consequence, the protein moves steadily 
forward (Figure 4-46). 

Many motor proteins generate directional movement by using the 
hydrolysis of a tightly bound ATP molecule to drive an orderly series of 
conformational changes. These movements can be rapid: the muscle 
motor protein myosin walks along actin filaments at about 6 ¡im/sec dur- 
ing muscle contraction (as discussed in Chapter 17). 

Proteins Often Form Large Complexes That Function as 
Protein Machines 

As one progresses from small, single-domain proteins to large proteins 
formed from many domains, the functions that the proteins can perform 
become more elabórate. The most complex tasks, however, are carried 
out by large protein assemblies formed from many protein molecules. 
Now that it is possible to reconstruct biological processes in cell-free Sys¬ 
tems in a test tube, it is clear that each central process in a cell—including 
DNA replication, gene transcription, protein synthesis, vesicle budding, 
and transmembrane signaling—is catalyzed by a highly coordinated, 
linked set of many proteins. In most such protein machines, the hydrol¬ 
ysis of bound nucleoside triphosphates (ATP or GTP) drives an ordered 
series of conformational changes in some of the individual protein subu- 
nits, enabling the ensemble of proteins to move coordinately. In this way, 
the appropriate enzymes can be positioned to carry out successive reac- 
tions in a series—as during the synthesis of proteins on a ribosome, for 
example (discussed in Chapter 7). Likewise, a large multiprotein complex 
moves rapidly along DNA to replícate the DNA double helix during cell 
división (discussed in Chapter 6). A simple mechanical analogy is illus- 
trated in Figure 4-47. 

Cells have evolved a large number of different protein machines suited to 
performing a variety of biological tasks. Cells employ protein machines 
for the same reason that humans have invented mechanical and elec- 
tronic machines: for almost any job, manipulations that are spatially and 
temporally coordinated through linked processes are much more efficient 
than is the sequential use of individual tools. 

Figure 4-46 A schematic model of how a motor protein uses ATP 
hydrolysis to move in one direction along a cytoskeletal filament. An 

orderly transltion among three conformations ¡s driven by the hydrolysis 
of a bound ATP molecule and the release of the producís: ADP and 
¡norganlc phosphate (P¡). Because these transltlons are coupled to the 
hydrolysis of ATP, the entire cycle is essentially irreversible. Through 
repeated cycles, the protein moves continuously to the right along 
the filament. The movement of a single molecule of myosin has been 
captured by atomic forcé microscopy. 

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How Proteins Are Studied 


Figure 4-47 "Protein machines" can carry out complex functions. These 
machines are made of individual proteins that collaborate to perform a specific 
task (Movie 4.11). The movement of these proteins is often coordinated by the 
hydrolysis of a bound nucleotide such as ATP. Conformational changes of this type 
are especially useful to the cell ¡f they occur ¡n a large protein assembly in which 
the activities of several different protein molecules can be coordinated by the 
movements within the complex. 


Understanding how a particular protein functions calis for detailed struc- 
tural and biochemical analyses—both of which require large amounts of 
puré protein. But isolating a single type of protein from the thousands 
of other proteins present in a cell is a formidable task. For many years, 
proteins had to be purified directly from the source—the tissues in which 
they are most plentiful. That approach was inconvenient, entailing, for 
example, early-moming trips to the slaughterhouse. More important, the 
complexity of intact tissues and organs is a major disadvantage when 
tiying to purify particular molecules, because a long series of chromatog- 
raphy steps is generally required. These procedures not only take weeks 
to perform, but they also yield only a few milligrams of puré protein. 
Nowadays, proteins are more often isolated from cells that are grown in 
a laboratoiy (see, for example, Figure 1-38). Often these cells have been 
"tricked" into making large quantities of a given protein using the genetic 
engineering techniques that we describe in Chapter 10. Such engineered 
cells frequently allow large amounts of puré protein to be obtained in 
only a few days. 

In this section, we outline how proteins are extracted and purified from 
cultured cells and other sources. We describe how these proteins are 
analyzed to determine their amino acid sequence and their three-dimen- 
sional structure. Finally, we discuss how technical advances are allowing 
proteins to be analyzed, cataloged, manipulated, and even designed from 


Explain why the hypothetical 
enzymes in Figure 4—47 have a 
great advantage in opening the 
safe if they work together in a 
protein complex, as opposed to 
working individually in an unlinked, 
sequential manner. 

Proteins Can be Purified from Cells or Tissues 

Whether starting with a piece of liver, a dish of cultured cells, or a vat 
of bacterial, yeast, or animal cells that have been engineered to pro¬ 
duce a protein of interest, the first step in any purification procedure is 
to break open the cells to release their contents. The resulting slurry is 
called a cell homogenate or extract. This physical disruption is followed by 
an initial fractionation procedure to sepárate out the class of molecules 
of interest—for example, all the soluble proteins in the cell (Panel 4-3, 
pp. 164-165). 

With this collection of proteins in hand, the job is then to isolate the 
desired protein. The standard approach involves purifying the protein 

LibertadDigital \ 2015 


CHAPTER 4 Protein Structure and Function 

protein X covalently 
attached to 
column matrix 

adhere to column 

IN pH 

purified X-binding proteins 

Figure 4-48 Affinity chromatography can be used to ¡solate the 
binding partners of a protein of interest. The purified protein 
of interest (protein X) is covalently attached to the matrix of a 
chromatography column. An extract containing a mixture of proteins 
is then loaded onto the column. Those proteins that associate with 
protein X inside the cell will usually bind to it on the column. Proteins 
not bound to the column pass right through, and the proteins that are 
bound tightly to protein X can then be released by changing the pH or 
ionic composition of the washing solution. 

through a series of chromatography steps, which use different materials 
to sepárate the individual components of a complex mixture into por- 
tions, or ftactions, based on the properties of the protein—such as size, 
shape, or electrical charge. After each separation step, the fractions are 
examined to determine which ones contain the protein of interest. These 
fractions are then pooled and subjected to additional chromatography 
steps until the desired protein is obtained in puré form. 

The most efficient forms of protein chromatography sepárate polypeptides 
on the basis of their ability to bind to a particular molecule—a process 
called affinity chromatography (Panel 4-4, p. 166). If large amounts of 
antibodies that recognize the protein are available, for example, they can 
be attached to the matrix of a chromatography column and used to help 
extract the protein frorn a mixture (see Panel 4-2, pp. 146-147). 

Affinity chromatography can also be used to isolate proteins that interact 
physically with the protein being studied. In this case, a purified protein 
of interest is attached tightly to the column matrix; the proteins that bind 
to it will remain in the column and can then be removed by changing the 
composition of the washing solution (Figure 4-48). 

Proteins can also be separated by electrophoresis. In this technique, a 
mixture of proteins is loaded onto a polymer gel and subjected to an 
electric field; the polypeptides will then migrate through the gel at differ¬ 
ent speeds depending on their size and net charge (Panel 4-5, p. 167). If 
too many proteins are present in the sample, or if the proteins are veiy 
similar in their migration rate, they can be resolved further using two- 
dimensional gel electrophoresis (see Panel 4-5). These electrophoretic 
approaches yield a number of bands or spots that can be visualized by 
staining; each band or spot contains a different protein. Chromatography 
and electrophoresis—both developed more than 50 years ago but greatly 
improved since—have been instrumental in building an understanding 
of what proteins look like and how they behave (Table 4-2). Both tech- 
niques are still frequently used in laboratories. 

Once a protein has been obtained in puré form, it can be used in bio- 
chemical assays to study the details of its activity. It can also be subjected 
to techniques that reveal its amino acid sequence and precise three- 
dimensional structure. 

Determining a Protein's Structure Begins with 
Determining Its Amino Acid Sequence 

The task of determining the amino acid sequence of a protein can be 
accomplished in several ways. For many years, sequencing a protein was 
done by directly analyzing the amino acids in the purified protein. First, 
the protein was broken down into smaller pieces using a selective pro- 
tease; the enzyme trypsin, for example, cleaves polypeptide chains on 
the carboxyl side of a lysine or an arginine. Then the identities of the 
amino acids in each fragment were determined chemically. The first pro¬ 
tein sequenced in this way was the hormone insulin, in 1955. 

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How Proteins Are Studied 




The ñame "protein" (from the Greek proteios, "primary") was suggested by Berzelius for the complex nitrogen-rich 
substance found ¡n the cells of all animáis and plants. 


Most of the 20 common amino acids found in proteins were discovered. 


Hoppe-Seyler crystallized, and named, the protein hemoglobin. 


Fischer proposed a lock-and-key analogy for enzyme-substrate ¡nteractions. 


Buchner and Buchner showed that cell-free extracts of yeast can break down sucrose to form carbón dioxide and 
ethanol, thereby laying the foundations of enzymology. 


Sumner crystallized urease in puré form, demonstrating that proteins could possess the catalytic activity of 
enzymes; Svedberg developed the first analytical ultracentrifuge and used itto estímate the correct molecular 
weight of hemoglobin. 


Tiselius ¡ntroduced electrophoresis for separating proteins in solution. 


Bernal and Crowfoot presented the first detailed X-ray diffraction patterns of a protein, obtained from crystals of 
the enzyme pepsin. 


Martin and Synge developed chromatography, a technique now widely used to sepárate proteins. 


Pauling and Corey proposed the structure of a helical conformation of a chain of amino acids—the a helix—and the 
structure of the P sheet, both of which were later found in many proteins. 


Sanger determined the order of amino acids in insulin, the first protein whose amino acid sequence was 


Ingram produced the first protein fingerprints, showing that the difference between sickle-cell hemoglobin and 
normal hemoglobin is due to a change in a single amino acid (Movie 4.12). 


Kendrew described the first detailed three-dimensional structure of a protein (sperm whale myoglobin) to a 
resolution of 0.2 nm, and Perutz proposed a lower-resolution structure for hemoglobin. 


Monod, Jacob, and Changeux recognized that many enzymes are regulated through allosteric changes in their 


Phillips described the three-dimensional structure of lysozyme by X-ray crystallography, the first enzyme to be 
analyzed in atomic detail. 


Nomura reconstituted a functional bacterial ribosome from purified components. 


Henderson and Unwin determined the first three-dimensional structure of a transmembrane protein 
(bacteriorhodopsin), using a computer-based reconstruction from electrón micrographs. 


Neher and Sakmann developed patch-clamp recording to measure the activity of single ¡on-channel proteins. 


Wüthrich used nuclear magnetic resonance (NMR) spectroscopy to solve the three-dimensional structure of a 
soluble sperm protein. 


Tanaka and Fenn separately developed methods for the analysis of proteins and other biological macromolecules. 


Mann, Aebersold, Yates, and others developed efficient methods for using mass spectrometry to identify proteins 
in complex mixtures, exploiting the availability of complete genome sequences. 

A much faster way to determine the amino acid sequence of proteins that 
have been isolated from organisms for which the full genome sequence is 
known is a method called mass spectrometry. This technique determines 
the exact mass of every peptide fragment in a purified protein, which then 
allows the protein to be identified from a database that contains a list of 
every protein thought to be encoded by the genome of the organism in 
question. Such lists are computed by taking the genome sequence of the 
organism and applying the genetic code (discussed in Chapter 7). 

To perform mass spectrometry, the peptides derived from digestión with 
trypsin are blasted with a láser. This treatment heats the peptides, caus- 
ing them to become electrically charged (ionized) and ejected in the form 
of a gas. Accelerated by a powerful electric field, the peptide ions then fly 
toward a detector; the time it takes them to arrive is related to their mass 
and their charge. (The larger the peptide is, the more slowly it moves; the 

LibertadDigital \ 2015 


CHAPTER 4 Protein Structure and Function 

single protein spot excised from gel 

Figure 4-49 Mass spectrometry can be used to identify proteins 
by determining the precise masses of peptides derived from 
them. As indicated, this ¡n turn allows the proteins to be produced in 
the large amounts needed for determining their three-dimensional 
structure. In this example, the protein of interest ¡s excised from a 
polyacrylamide gel aftertwo-dimensional electrophoresis (see Panel 
4-5, p. 167) and then digested with trypsin. The peptide fragments 
are loaded into the mass spectrometer, and their exact masses are 
measured. Genome sequence databases are then searched to find the 
protein encoded by the organism in question whose profile matches 
this peptide fingerprint. Mixtures of proteins can also be analyzed in 
this way. (Image courtesy of Patrick O'Farrell.) 



y- (mass to charge ratio) 






more highly charged it is, the faster it moves.) The set of exact masses of 
the protein fragments produced by trypsin cleavage then serves as a "fin¬ 
gerprint" that identifles the protein—and its corresponding gene—from 
publicly accessible databases (Figure 4-49). 

This approach can even be applied to complex mixtures of proteins, 
for example, starting with an extract containing all the proteins made 
by yeast cells grown under a particular set of conditions. To obtain the 
increased resolution required to distinguish individual proteins, such 
mixtures are frequently analyzed using tándem mass spectrometry. In this 
case, after the peptides pass through the flrst mass spectrometer, they 
are broken into even smaller fragments and analyzed by a second mass 

Although all the information required for a polypeptide chain to fold is 
contained in its amino acid sequence, we have not yet learned how to 
reliably predict a protein's detailed three-dimensional conformation—the 
spatial arrangement of its atoms—from its sequence alone. At present, 
the only way to discover the precise folding pattem of any protein is by 
experiment, using either X-ray ciystallography or nuclear magnetic 
resonance (NMR) spectroscopy (How We Know, pp. 162-163). 

Genetic Engineering Techniques Permit the Large-Scale 
Production, Design, and Analysis of Almost Any Protein 

Advances in genetic engineering techniques now permit the production 
of large quantities of almost any desired protein. In addition to making 
life much easier for biochemists interested in purifying specific proteins, 
this ability to chum out huge quantities of a protein has given rise to an 
entire biotechnology industry (Figure 4-50). Bacteria, yeast, and cultured 
mammalian cells are now used to mass produce a variety of therapeutic 
proteins, such as insulin, human growth hormone, and even the fertility- 
enhancing drugs used to boost egg production in women undergoing in 
vitro fertilization. Preparing these proteins previously required the col- 
lection and processing of vast amounts of tissue and other biological 
producís—including, in the case of the fertility drugs, the uriñe of post- 
menopausal nuns. 

The same sorts of genetic engineering techniques can also be employed 
to produce new proteins and enzymes that contain novel structures or 
perform unusual tasks: metabolizing toxic wastes, synthesizing life- 
saving drugs, or operating under conditions that would destroy most 
biological catalysts (see Chapter 3 How We Know, pp. 104-106). Most 
of these synthetic catalysts are nowhere near as effective as naturally 
occurring enzymes in terms of their ability to speed the rate of selected 
Chemical reactions. But, as we continué to learn more about how pro¬ 
teins and enzymes exploit their unique conformations to carry out their 
biological functions, our ability to make novel proteins with useful func- 
tions can only improve. 

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How Proteins Are Studied 


Of course, to be able to study—or benefit from—the activity of an engi- 
neered protein in a living organism, the DNA encoding that protein must 
somehow be introduced into cells. Again, thanks to genetic engineering 
techniques, we are able to do just that. We discuss these methods in great 
detail in Chapter 10. 

The Relatedness of Proteins Aids the Prediction of Protein 
Structure and Function 

Biochemists have made enormous progress in understanding the struc¬ 
ture and function of proteins over the past 150 years (see Table 4-2, 
p. 159). These advances are the fruits of decades of painstaking research 
on isolated proteins, performed by individual scientists working tirelessly 
on single proteins or protein families, one by one, sometimes for their 
entire careers. In the future, however, more and more of these investiga- 
tions of protein conformation and activity will likely take place on a larger 

Improvements in our ability to rapidly sequence whole genomes, and 
the development of methods such as mass spectrometiy, have fueled 
our ability to determine the amino acid sequences of enormous num- 
bers of proteins. Millions of unique protein sequences from thousands 
of different species have thereby been deposited into publicly avail- 
able databases, and the collection is expected to double in size eveiy 
two years. Comparing the amino acid sequences of all of these proteins 
reveáis that the majority belong to protein families that share specific 
"sequence patterns"—stretches of amino acids that fold into distinct 
structural domains. In some of these families, the proteins contain only a 
single structural domain. In others, the proteins inelude múltiple domains 
arranged in novel combinations (Figure 4-51). 

Although the number of multidomain families is growing rapidly, the 
discovery of novel single domains appears to be leveling off. This pla- 
teau suggests that the vast majority of proteins may fold up into a limited 
number of structural domains—perhaps as few as 10,000 to 20,000. For 
many single-domain families, the structure of at least one family member 
is known. And knowing the structure of one family member allows us 
to say something about the structure of its relatives. By this account, we 
have some structural information for almost three-quarters of the pro¬ 
teins archived in databases (Movie 4.13). 

A future goal is to acquire the ability to look at a protein’s amino acid 
sequence and be able to deduce its structure and gain insight into its 
function. We are coming closer to being able to predict protein struc¬ 
ture based on sequence information, but there is still a long way to go. 
Predicting how a protein will function, alone, as part of a complex, or as 
part of a network in the cell, is much more challenging. But, the closer we 
get to addressing these questions, the closer we should be to understand¬ 
ing the fundamental basis of life. 

Figure 4-51 Most proteins belong to structurally related families. 

(A) More than two-thirds of all well-studied proteins contain a single 
structural domain. The members of these single-domain families 
can have different amino acid sequences but fold into a protein 
with a similar shape. (B) During evolution, structural domains have 
been combined in different ways to produce families of multidomain 
proteins. Almost all novelty in protein structure comes from the way 
these single domains are arranged. The number of multidomain 
families being added to the public databases is still rapidly increasing, 
unlike the number of novel single domains. 

Figure 4-50 Biotechnology companies 
produce mass quantities of useful 
proteins. Shown in this photograph are 
the fermenters used to grow the cells 
needed for such large-scale protein 
production. (Courtesy of Bioengineering 
AG, Switzerland.) 

family 1 family 2 

(A) single-domain protein families 


(B) a two-domain protein family 

LibertadDigital \ 2015 




As you've no doubt already concluded in reading this 
chapter, for many proteins, their three-dimensional 
shape determines their function. So to leam more about 
how a protein works, it helps to know exactly what it 
looks like. 

The problem is that most proteins are too small to be 
seen in any detail, even with a powerful electrón micro- 
scope. To follow the path of an amino acid chain that 
is folded into a protein molecule, you need to be able 
to "see" its individual atoms. Scientists use two main 
methods to map the locations of atoms in a protein. 
The first involves the use of X-rays. Like light, X-rays 
are a form of electromagnetic radiation. But they have a 
wavelength that's much shorter: 0.1 nanometer (nm) as 
opposed to the 400-700 nm wavelength of visible light. 
That tiny wavelength—which is the approximate diam- 
eter of a hydrogen atom—allows scientists to probe the 
structure of very small objects at the atomic level. 

A second method, called nuclear magnetic resonance 
(NMR) spectroscopy, takes advantage of the fact that— 
in many atoms—the nucleus is intrinsically magnetic. 
When exposed to a large magnet, these nuclei act like 
tiny bar magnets and align themselves with the mag¬ 
netic field. If they are then excited with a blast of radio 
waves, the nuclei will wobble around their magnetic 
axes, and, as they relax back into the aligned position, 
they will give off a signal that can be used to reveal their 
relative positions in a protein. 

Using these techniques, investigators have painstakingly 
pieced together many thousands of protein structures. 
With the help of Computer graphics programs, they have 
been able to traverse the surfaces and climb inside these 
proteins, exploring the nooks where ATP likes to nes- 
tle, for example, or examining the loops and hélices that 
proteins use to grab hold of a ligand or wrap around a 
segment of DNA. If the protein happens to belong to a 
virus or to a cáncer cell, seeing its structure can provide 
clues to designing drugs that might thwart an infection 
or elimínate a tumor. 


To determine a protein's structure using X-ray crystal- 
lography, you first need to coax the protein into forming 
ciystals: large, highly ordered arrays of the puré protein 
in which every molecule has the same conformation and 
is perfectly aligned with its neighbors. Growing high- 
quality protein crystals is still something of an art and 
is largely a matter of trial and error. Although robotic 
methods increase efficiency, it can still take years to find 
the right conditions—and some proteins resist crystal- 
lization altogether. 

If you're lucky enough to get good crystals, you are 
ready for the X-ray analysis. When a narrow beam of 
X-rays is directed at a protein crystal, the atoms in the 
protein molecules scatter the incoming X-rays. These 
scattered waves either reinforce or cancel one another, 
producing a complex diffraction pattern that is collected 
by electronic detectors. The position and intensity 
of each spot in the diffraction pattern contains infor- 
mation about the position of the atoms in the protein 
crystal (Figure 4-52). 

Because these pattems are so complex—even a small 
protein can generate 25,000 discrete spots—computers 
are used to interpret them and transform them by com¬ 
plex mathematical calculations into maps of the relative 
spatial positions of the atoms. By combining information 
obtained from such maps with the amino acid sequence 
of the protein, you can eventually generate an atomic 
model of the protein's structure. To determine whether 
the protein undergoes conformational changes in its 
structure when it binds a ligand that boosts its activity, 
you might subsequently try ciystallizing it in the pres- 
ence of its ligand. With crystals of sufficient quality, even 
small atomic movements can be detected by comparing 
the structures obtained in the presence and absence of 
stimulatory or inhibitory ligands. 


The trouble with X-ray crystallography is that you need 
crystals. And not all proteins like to form such orderly 
assemblies. Many have intrinsically disordered regions 
that wiggle around too much to stack neatly into a ciys- 
talline array. Others might not ciystallize in the absence 
of the membranes in which they normally reside. 

The other way to solve the structure of a protein does 
not require protein crystals. If the protein is small—say, 
50,000 daltons or less—you can determine its structure 
by NMR spectroscopy. In this technique, a concentrated 
solution of puré protein is placed in a strong magnetic 
field and then bombarded with radio waves of different 
frequencies. Hydrogen nuclei, in particular, will gener¬ 
ate an NMR signal that can be used to determine the 
distances between these atoms in different parts of the 
protein. This information is then used to build a model 
of how the hydrogens are arranged in space. Again, 
combined with the known amino acid sequence, an 
NMR spectrum can allow you to compute the three- 
dimensional structure of the protein (Figure 4-53). If 
the protein is larger than 50,000 daltons, you can try to 
break it up into its constituent functional domains and 
analyze each domain by NMR. 

LibertadDigital \ 2015 

How Proteins Are Studied 


Figure 4-52 The structure of a protein can be determined by X-ray crystallography. Ribulose bisphosphate carboxylase is an 
enzyme that plays a central role ¡n CO2 fixatlon durlng photosynthesis. (A) X-ray diffraction apparatus; (B) photograph of crystal; 

(C) diffraction pattern; (D) three-dimensional structure determined from the pattern (a hélices are shown in green, and P sheets ¡n red). 
(B, courtesy of C. Branden; C, courtesy of J. Hajdu and I. Anderson; D, adapted from original provided by B. Furugren.) 

Because determining the precise conformation of a pro¬ 
tein is so time-consuming and costly—and the resulting 
insights so valuable—scientists routinely make their 
structures freely available by submitting the informa- 
tion to a publicly accessible database. Thanks to such 
databases, anyone interested in viewing the structure 
of, say, the ribosome—a complex macromolecular 
machine made of several RNAs and more than 50 pro¬ 
teins—can easily do so. In the future, improvements in 

X-ray crystallography and NMR spectroscopy should 
permit rapid analysis of many more proteins and pro¬ 
tein machines. And once enough structures have been 
determined, it might become possible to generate algo- 
rithms for accurately predicting structure solely on the 
basis of a protein's amino acid sequence. After all, it is 
the sequence of the amino acids alone that determines 
how each protein folds up into its three-dimensional 

Figure 4-53 NMR spectroscopy can be used 
to determine the structure of small proteins 
or protein domains. (A) Two-dimensional NMR 
spectrum derived from the C-terminal domain 
of the enzyme cellulase, which breaks down 
cellulose. The spots represent interactions between 
neighboring hydrogen atoms. (B) The set of 
overlapping structures shown all satisfy the distance 
constraints equally well. (Courtesy of P. Kraulis.) 

bertadDigital \ 2015 



How Proteins Are Studied 




Proteins are very diverse. They differ in 
size, shape, charge, hydrophobicity, and 
their affinity for other molecules. All of 
these properties can be exploited to 
sepárate them from one another so 
that they can be studied individually. 


Although the material used to form 
the matrix for column chromatography 
varíes, ¡t is usually packed in the 
column in the form of small beads. 
Atypical protein purification strategy 
might employ in turn each of the 
three kinds of matrix described 
below, with a final protein 
purification of up to 10,000-fold. 

Purity can easily be assessed by gel 
electrophoresis (Panel 4-5). 


Proteins are often fractionated by column chromatography. A mixture of proteins in 
solution is applied to the top of a cylindrical column filled with a permeable solid 
matrix immersed in solvent. A large amount of solvent is then pumped through the 
column. Because different proteins are retarded to different extents by their 
interaction with the matrix, they can be collected separately as they flow out from 
the bottom. According to the choice of matrix, proteins can be separated according 
to their charge, hydrophobicity, size, or ability to bind to particular Chemical 
groups (see below). 

solvent continuously 
applied to the top of 
sample column from a large 
applied reservoir of solvent 

solvent flow 

solvent flow 

solvent flow 


I I I 

+ posltively 
i—— charged 
+ + # bead 

+ bound 
* negatively 


+ # + molecule 

®.- positively 



. * # • 

• •' 

large molecules 

bead with 








other proteins 
pass through 


lon-exchange columns are packed with 
small beads carrying either positive or 
negative charges that retard proteins 
of the opposite charge. The association 
between a protein and the matrix 
depends on the pH and ionic strength 
of the solution passing down the 
column. These can be varied in a 
controlled way to achieve an effective 


Gel-filtration columns sepárate 
proteins according to their size. The 
matrix consists of tiny porous beads. 
Protein molecules that are small 
enough to enter the holes in the beads 
are delayed and travel more slowly 
through the column. Proteins that 
cannot enter the beads are washed out 
of the column first. Such columns also 
allow an estímate of protein size. 


Affinity columns contain a matrix 
covalently coupled to a molecule that 
interacts specifically with the protein 
of ¡nterest (e.g., an antibody, or an 
enzyme substrate). Proteins that bind 
specifically to such a column can 
subsequently be released by a pH 
change or by concentrated salt 
Solutions, and they emerge highly 
purified (see also Figure 4-48). 






The detergent CH3 

sodium dodecyl 
sulfate (SDS) Chb 

solubilize CH2 

proteins for SDS 
polyacrylamide- CH2 

gel electrophoresis. | 

CH 2 


protein with two 
subunits, A and B, 
joined by a disulfide 
(S—S) bond 


When an electric field is applied to a solution 
containing protein molecules, the molecules 
will mígrate in a direction and at a speed that 
reflects their size and net charge. This forms 
the basis of the technique called 


For any protein there is a characteristic 
pH, called the isoelectric point, at which 
the protein has no net charge and 
therefore will not move in an electric 
field. In isoelectric focusing, proteins 
are electrophoresed in a narrow tube of 
polyacrylamide gel in which a pH 
gradient is established by a mixture of 
special buffers. Each protein movesto a 
point in the pH gradient that corresponds 
to its isoelectric point and stays there. 

stable pH gradient 


The protein shown h< 

in Isoelectric pH of 6.5. 


¿0 Na© 

SDS polyacrylamide-gel electrophoresis B 


Individual polypeptide chains form a complex with q 
negatively charged molecules of sodium dodecyl 
sulfate (SDS) and therefore migrate as negatively 
charged SDS-protein complexes through a slab of 
porous polyacrylamide gel. The apparatus used for A 
this electrophoresis technique is shown above ( left). 

A reducing agent (mercaptoethanol) is usually added 
to break any-S-S- linkages within or between 
proteins. Under these conditions, unfolded polypeptide 
chains migrate at a rate that reflects their molecular 


=Ir negatively 
charged SDS 


slab of polyacrylamide gel 


Complex mixtures of proteins cannot be resolved well on one-dimensional gels, but 
two-dimensional gel electrophoresis, combining two different separation methods, can 
be used to resolve more than 1000 proteins in a two-dimensional protein map. In the 
first step, native proteins are separated in a narrow gel on the basis of their intrinsic 
charge using isoelectric focusing (see left). In the second step, this gel is placed on top of 
a gel slab, and the proteins are subjected to SDS-PAGE (see above) in a direction 
perpendicular to that used in the first step. Each protein migrates to form a discrete spot. 

All the proteins in 

an E. coli bacterial basic -<-stable pH gradient-► acidic 

cell are separated _ — 

in this two- ^ ~ _ 

dimensional gel, in 100 -Y 1*. — sr 

which each spot ^ ♦ 

corresponds to a n 


polypeptide chain. | 50 

They are separated c 

according to their 
isoelectric point 01 

from left to right E 25 

and to their q 

molecular weight . 

from top to y 

bottom. (Courtesy 
of Patrick O'Farrell.) 

v -- a -i 

-- ."Y : - - 



Protein Structure and Function 


• Living cells contain an enormously diverse set of protein molecules, 
each made as a linear chain of amino acids linked together by cova- 
lent peptide bonds. 

• Each type of protein has a unique amino acid sequence, which deter¬ 
mines both its three-dimensional shape and its biological activity. 

• The folded structure of a protein is stabilized by múltiple noncovalent 
interactions between different parts of the polypeptide chain. 

• Hydrogen bonds between neighboring regions of the polypeptide 
backbone often give rise to regular folding patterns, known as a héli¬ 
ces and p sheets. 

• The structure of many proteins can be subdivided into smaller globu¬ 
lar regions of compact three-dimensional structure, known as protein 

• The biological function of a protein depends on the detailed Chemical 
properties of its surface and how it binds to other molecules, called 

• When a protein catalyzes the formation or breakage of a specific 
covalent bond in a ligand, the protein is called an enzyme and the 
ligand is called a substrate. 

• At the active site of an enzyme, the amino acid side chains of the 
folded protein are precisely positioned so that they favor the for¬ 
mation of the high-energy transition States that the substrates must 
pass through to be converted to product. 

• The three-dimensional structure of many proteins has evolved so 
that the binding of a small ligand can induce a significant change in 
protein shape. 

• Most enzymes are allosteric proteins that can exist in two conforma- 
tions that differ in catalytic activity, and the enzyme can be tumed 
on or off by ligands that bind to a distinct regulatory site to stabilize 
either the active or the inactive conformation. 

• The activities of most enzymes within the cell are strictly regulated. 
One of the most common forms of regulation is feedback inhibition, 
in which an enzyme early in a metabolic pathway is inhibited by the 
binding of one of the pathway's end products. 

• Many thousands of proteins in a typical eukaryotic cell are regulated 
by cycles of phosphorylation and dephosphorylation. 

• GTP-binding proteins also regúlate protein function in eukaryotes; 
they act as molecular switches that are active when GTP is bound 
and inactive when GDP is bound; tuming themselves off by hydrolyz- 
ing their bound GTP to GDP. 

• Motor proteins produce directed movement in eukaryotic cells 
through conformational changes linked to the hydrolysis of ATP to 

• Highly efficient protein machines are formed by assemblies of allos¬ 
teric proteins in which the various conformational changes are 
coordinated to perform complex functions. 

• Covalent modifications added to a protein's amino acid side chains 
can control the location and function of the protein and can serve as 
docking sites for other proteins. 

• Starting from crude cell or tissue homogenates, individual proteins 
can be obtained in puré form by using a series of chromatography 

• The function of a purified protein can be discovered by biochemical 
analyses, and its exact three-dimensional structure can be deter- 
mined by X-ray crystallography or NMR spectroscopy. 

LibertadDigital \ 2015 

Chapter 4 End-of-Chapter Questions 



active site 

mass spectrometry 


motor protein 

a helix 


amino acid sequence 

nuclear magnetic resonance 


(NMR) spectroscopy 


peptide bond 

P sheet 

polypeptide, polypeptide chain 

binding site 

polypeptide backbone 


primary structure 




protein domain 


protein family 

disulfide bond 

protein kinase 


protein machine 


protein phosphatase 

feedback inhibition 

protein phosphorylation 

fibrous protein 

quaternary structure 

globular protein 

secondary structure 

GTP-binding protein 

side chain 



intrinsically disordered 



tertiary structure 


transition State 


X-ray crystallography 



Look at the models of the protein ¡n Figure 4-12. Is the 
red a helix right- or left-handed? Are the three strands that 
form the large (3 sheet parallel or antiparallel? Starting at 
the N-terminus (the purple end), trace your finger along the 
peptide backbone. Are there any knots? Why, or why not? 


Which of the following statements are corred? Explain your 

A. The adive site of an enzyme usually occupies only a 
small fradion of the enzyme surface. 

B. Catalysis by some enzymes involves the formation of 
a covalent bond between an amino acid side chain and a 
substrate molecule. 

C. A P sheet can contain up to five strands, but no more. 

D. The specificity of an antibody molecule is contained 
exclusively in loops on the surface of the folded light-chain 

E. The possible linear arrangements of amino acids are so 
vast that new proteins almost never evolve by alteration of 
oíd ones. 

F. Allosteric enzymes have two or more binding sites. 

G. Noncovalent bonds are too weak to influence the three- 
dimensional strudure of macromolecules. 

H. Affinity chromatography separates molecules according 
to their intrinsic charge. 

I. Upon centrifugation of a cell homogenate, smaller 
organelles experience less fridion and thereby sediment 
faster than larger ones. 


What common feature of a hélices and P sheets makes them 
universal building blocks for proteins? 


Protein strudure is determined solely by a protein's amino 
acid sequence. Should a genetically engineered protein in 
which the original order of all amino acids is reversed have 
the same strudure as the original protein? 


Consider the following protein sequence as an a helix: 

How many turns does this helix make? Do you find anything 
remarkable about the arrangement of the amino acids in 
this sequence when folded into an a helix? (Hint: consult the 
properties of the amino acids in Figure 4-3.) 


Simple enzyme readions often conform to the equation 
E + S^ES^EP^E + P 

LibertadDigital \ 2015 

170 CHAPTER 4 Protein Structure and Function 

where E, S, and P are enzyme, substrate, and product, 

A. What does ES represent ¡n this equation? 

B. Why is the first step shown with bidirectional arrows and 
the second step as a unidirectional arrow? 

C. Why does E appear at both ends of the equation? 

D. One often finds that high concentrations of P inhibit the 
enzyme. Suggest why this might occur. 

E. If compound X resembles S and binds to the active site 
of the enzyme but cannot undergo the reaction catalyzed 
by it, what effects would you expect the addition of X to 
the reaction to have? Compare the effects of X and of the 
accumulation of P. 


Which of the following amino acids would you expect 
to find more often near the center of a folded globular 
protein? Which ones would you expect to find more often 
exposed to the outside? Explain your answers. Ser, Ser-P (a 
Ser residue that is phosphorylated). Leu, Lys, Gln, His, Phe, 
Val, lie, Met, Cys-S-S-Cys (two cysteines that are disulfide- 
bonded), and Glu. Where would you expect to find the most 
N-terminal amino acid and the most C-terminal amino acid? 


Assume you want to make and study fragments of a protein. 
Would you expect that any fragment of the polypeptide 
chain would fold the same way as it would in the intact 
protein? Consider the protein shown in Figure 4-19. Which 
fragments do you suppose are most likely to fold correctly? 


Neurofilament proteins assemble into long, intermedíate 
filaments (discussed in Chapter 17), found in abundance 
running along the length of nerve cell axons. The C-terminal 
región of these proteins is an unstructured polypeptide, 
hundreds of amino acids long and heavily modified by the 
addition of phosphate groups. The term "polymer brush" 
has been applied to this part of the neurofilament. Can you 
suggest why? 


An enzyme isolated from a mutant bacterium grown at 
20°C works in a test tube at 20°C but not at 37°C (37°C is 
the temperature of the gut, where this bacterium normally 
lives). Furthermore, once the enzyme has been exposed 
to the higher temperature, it no longer works at the lower 
one. The same enzyme isolated from the normal bacterium 
works at both temperatures. Can you suggest what happens 
(at the molecular level) to the mutant enzyme as the 
temperature increases? 


A motor protein moves along protein filaments in the cell. 
Why are the elements shown in the illustration not sufficient 
to medíate directed movement (Figure Q4-19)? With 
reference to Figure 4-46, modify the illustration shown 
here to inelude other elements that are required to create a 
unidirectional motor, and justify each modification you make 
to the illustration. 

Figure Q4-19 


Gel-filtration chromatography separates molecules 
according to their size (see Panel 4-4, p. 166). Smaller 
molecules diffuse faster in solution than larger ones, yet 
smaller molecules migrate more slowly through a gel- 
filtration column than larger ones. Explain this paradox. 
What should happen at very rapid flow rates? 


As shown in Figure 4-16, both a hélices and the coiled-coil 
structures that can form from them are helical structures, 
but do they have the same handedness in the figure? 
Explain why? 


How is it possible for a change in a single amino acid in a 
protein of 1000 amino acids to destroy its function, even 
when that amino acid is far away from any ligand-binding 

LibertadDigital \ 2015 



DNA and Chromosomes 

Life depends on the ability of cells to store, retrieve, and transíate the 
genetic instructions required to make and maintain a living organism. 
This hereditary information is passed on from a cell to its daughter cells 
at cell división, and from generation to generation in multicellular organ- 
isms through the reproductive cells—eggs and sperm. These instructions 
are stored within every living cell in its genes —the information-contain- 
ing elements that determine the characteristics of a species as a whole 
and of the individuáis within it. 

At the beginning of the twentieth century, when genetics emerged as 
a Science, scientists became intrigued by the Chemical nature of genes. 
The information in genes is copied and transmitted from cell to daughter 
cells millions of times during the life of a multicellular organism, and it 
survives the process essentially unchanged. What kind of molecule could 
be capable of such accurate and almost unlimited replication, and also be 
able to direct the development of an organism and the daily life of a cell? 
What kind of instructions does the genetic information contain? How are 
these instructions physically organized so that the enormous amount of 
information required for the development and maintenance of even the 
simplest organism can be contained within the tiny space of a cell? 

The answers to some of these questions began to emerge in the 1940s, 
when it was discovered from studies in simple fungí that genetic infor¬ 
mation consists primarily of instructions for making proteins. Proteins 
perform most of the cell's functions: they serve as building blocks for 
cell structures; they form the enzymes that catalyze the cell's Chemical 
reactions; they regúlate the activity of genes; and they enable cells to 




LibertadDigital \ 2015 

172 CHAPTER 5 DNA and Chromosomes 

dividing cell nondividing cell 

Figure 5-1 Chromosomes become visible 
as eukaryotic cells prepare to divide. 

(A) Two adjacent plant cells photographed 
¡n a fluorescence microscope. The DNA 
¡s labeled with a fluorescent dye (DAPI) 
that binds to ¡t. The DNA ¡s packaged 
into chromosomes, which become visible 
as distinct structures only when they 
condense in preparation for cell división, 
as shown on the left. The cell on the 
right, which ¡s not dividing, contains the 
¡dentical chromosomes, but they cannot be 
distinguished as individual entities because 
the DNA is in a much more extended 
conformation atthis phase in the cell's life 
cycle. (B) Schematic diagram of the outlines 
of the two cells and their chromosomes. 

(A, courtesy of Peter Shaw.) 

move and to communicate with one another. With hindsight, it is hard 
to imagine what other type of instructions the genetic information could 
have contained. 

The other crucial advance made in the 1940s was the recognition that 
deoxyribonucleic acid (DNA) was the likely carrier of this genetic informa¬ 
tion. But the mechanism whereby the hereditary information is copied for 
transmission frorn one generation of cells to the next, and how proteins 
are specified by the instructions in DNA, remained completely mysterious 
until 1953, when the structure of DNA was determined by James Watson 
and Francis Crick. The structure immediately revealed how DNA might be 
copied, or replicated, and it provided the first clues about how a molecule 
of DNA might encode the instructions for making proteins. Today, the fact 
that DNA is the genetic material is so fundamental to our understanding 
of life that it is difficult to appreciate what an enormous intellectual gap 
this discovery filled. 

In this chapter, we begin by describing the structure of DNA. We see how, 
despite its Chemical simplicity, the structure and Chemical properties of 
DNA make it ideally suited for carrying genetic information. The genes of 
every cell on Earth are made of DNA, and insights into the relationship 
between DNA and genes have come frorn experiments in a wide variety 
of organisms. We then consider how genes and other important segments 
of DNA are arranged in the single, long DNA molecule that forms the core 
of each chromosome in the cell. Finally, we discuss how eukaryotic cells 
fold these long DNA molecules into compact chromosomes inside the 
nucleus. This packing has to be done in an orderly fashion so that the 
chromosomes can be duplicated and apportioned correctly between the 
two daughter cells at each cell división. It must also allow the DNA to be 
accessed by the proteins that replícate and repair DNA, and regúlate the 
activity of its many genes. 

This is the first of five chapters that deal with basic genetic mechanisms— 
the ways in which the cell maintains and makes use of the genetic 
information carried in its DNA. In Chapter 6, we discuss the mechanisms 
by which the cell accurately replicates and repairs its DNA. In Chapter 7, 
we consider gene expression—how genes are used to produce RNA and 
protein molecules. In Chapter 8, we describe how a cell Controls gene 
expression to ensure that each of the many thousands of proteins encoded 
in its DNA is manufactured at the proper time and place. In Chapter 9, we 
discuss how present-day genes evolved frorn distant ancestors, and, in 
Chapter 10, we consider some of the experimental techniques used to 
study both DNA and its role in fundamental cell processes. 

An enormous amount has been learned about these subjects in the past 
60 years. Much less obvious, but equally important, is that our knowledge 
is very incomplete; thus a great deal still remains to be discovered about 
how DNA provides the instructions to build living things. 


Well before biologists understood the structure of DNA, they had rec- 
ognized that inherited traits and the genes that determine them were 
associated with the chromosomes. Chromosomes (named frorn the 
Greek chroma, "color," because of their staining properties) were discov¬ 
ered in the nineteenth century as threadlike structures in the nucleus of 
eukaryotic cells that become visible as the cells begin to divide (Figure 
5-1). As biochemical analysis became possible, researchers learned that 
chromosomes contain both DNA and protein. But which of these compo- 
nents encoded the organism's genetic information was not clear. 

LibertadDigital \ 2015 

The Structure of DNA 


We now know that the DNA carnes the hereditary information of the 
cell and that the protein components of chromosomes function largely to 
package and control the enormously long DNA molecules. But biologists 
in the 1940s had difficulty accepting DNA as the genetic material because 
of the apparent simplicity of its chemistiy (see How We Know, pp. 174— 
176). DNA, after all, is simply a long polymer composed of only four types 
of nucleotide subunits, which are chemically very similar to one another. 
Then, early in the 1950s, DNA was examined by X-ray diffraction analy- 
sis, a technique for determining the three-dimensional atomic structure 
of a molecule (see Figure 4-52). The early results indicated that DNA 
is composed of two strands wound into a helix. The observation that 
DNA is double-stranded was of crucial significance. It provided one of 
the major clues that led, in 1953, to a correct model for the structure 
of DNA. This structure immediately suggested how DNA could encode 
the instructions necessaiy for life, and how these instructions could be 
copied and passed along when cells divide. In this section, we examine 
the structure of DNA and explain in general terms how it is able to store 
hereditary information. 

A DNA Molecule Consists of Two Complementary Chains 
of Nucleotides 

A molecule of deoxyribonucleic acid (DNA) consists of two long poly- 
nucleotide chains. Each chain, or strand, is composed of four types of 
nucleotide subunits, and the two strands are held together by hydrogen 
bonds between the base portions of the nucleotides (Figure 5-2). 

(A) building blocks of DNA 
phosphate / 

'•m. + \ — 

sugar- base 

phosphate (guanine) 



s •TCTCKK 1 3 

G C A T ’ 

Figure 5-2 DNA is made of four 
nucleotide building blocks. (A) Each 
nucleotide is composed of a sugar- 
phosphate covalently linked to a 
base—guanine (G) in this figure. (B) The 
nucleotides are covalently linked together 
into polynucleotide chains, with a sugar- 
phosphate backbone from which the bases 
(A, C, G, and T) extend. (C) A DNA molecule 
is composed of two polynucleotide chains 
(DNA strands) held together by hydrogen 
bonds between the paired bases. The 
arrows on the DNA strands indícate the 
polarities of the two strands, which run 
antiparallel to each other in the DNA 
molecule. (D) Although the DNA is shown 
straightened out in (C), in reality, it is wound 
into a double helix, as shown here. 

LibertadDigital \ 2015 




By the 1920s, scientists generally agreed that genes 
reside on chromosomes, and they knew that chromo- 
somes are composed of both DNA and proteins. But 
because DNA is so chemically simple, they naturally 
assumed that genes had to be made of proteins, which 
are much more chemically diverse than DNA molecules. 
Even when the experimental evidence suggested other- 
wise, this assumption proved hard to shake. 

Messages from the dead 

The case for DNA began to emerge in the late 1920s, 
when a British medical officer named Fred Griffith made 
an astonishing discovery. He was studying Streptococcus 
pneumoniae (pneumococcus), a bacterium that causes 
pneumonía. As antibiotics had not yet been discovered, 
infection with this organism was usually fatal. When 

mouse dies 
of infection 

mouse lives 


mouse dies living, pathogenic 

of infection S strain recovered 

Figure 5-3 Griffith showed that 
heat-killed, infectious bacteria can 
transform harmless, living bacteria 
into pathogenic ones. The bacterium 
Streptococcus pneumoniae comes in two 
forms that differ from one another in their 
microscopio appearance and in their ability 
to cause disease. Cells of the pathogenic 
strain, which are lethal when ¡njected into 
mice, are encased in a slimy, glistening 
polysaccharide capsule. When grown on 
a píate of nutrients in the laboratory, this 
disease-causing bacterium forms colonies 
that look dome-shaped and smooth; 
henee it is designated the S form. The 
harmless strain of the pneumococcus, 
on the other hand, lacks this protective 
coat; it forms colonies that appear fíat 
and rough—henee, it is referred to as the 
R form. As ¡llustrated, Griffith found that 
a substance present in the pathogenic 
S strain could permanently change, or 
transform, the nonlethal R strain into the 
deadly S strain. 

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The Structure of DNA 


grown in the laboratory, pneumococci come in two 
forms: a pathogenic form that causes a lethal infection 
when injected into animáis, and a harmless form that is 
easily conquered by the animal’s immune System and 
does not produce an infection. 

In the course of his investigations, Grifflth injected vari- 
ous preparations of these bacteria into mice. He showed 
that pathogenic pneumococci that had been killed by 
heating were no longer able to cause infection. The 
surprise carne when Griffith injected both heat-killed 
pathogenic bacteria and live harmless bacteria into the 
same mouse. This combination proved lethal: not only 
did the animáis die of pneumonía, but Griffith found that 
their blood was teeming with live bacteria of the patho¬ 
genic form (Figure 5-3). The heat-killed pneumococci 
had somehow converted the harmless bacteria into the 
lethal form. What's more, Griffith found that the change 
was permanent: he could grow these "transformed" bac¬ 
teria in culture, and they remained pathogenic. But what 
was this mysterious material that turned harmless bac¬ 
teria into killers? And how was this change passed on to 
progeny bacteria? 


Griffith's remarkable finding set the stage for the experi- 
ments that would provide the first strong evidence that 
genes are made of DNA. The American bacteriologist 
Oswald Aveiy, following up on Griffith's work, discovered 
that the harmless pneumococcus could be transformed 
into a pathogenic strain in a culture tube by exposing 
it to an extract prepared from the pathogenic strain. It 
would take another 15 years, however, for Avery and 
his colleagues Colín MacLeod and Maclyn McCarty to 
successfully purify the "transforming principie" from 
this soluble extract and to demónstrate that the active 
ingredient was DNA. Because the transforming principie 
caused a heritable change in the bacteria that received 
it, DNA must be the very stuff of which genes are made. 
The 15-year delay was in part a reflection of the aca- 
demic climate—and the widespread supposition that 
the genetic material was likely to be made of protein. 
Because of the potential ramifications of their work, the 
researchers wanted to be absolutely certain that the 
transforming principie was DNA before they announced 
their findings. As Avery noted in a letter to his brother, 
also a bacteriologist, "It's lots of fun to blow bubbles, 
but it’s wiser to prick them yourself before someone else 
tries to." So the researchers subjected the transform¬ 
ing material to a battery of Chemical tests (Figure 5-4). 
They found that it exhibited all the Chemical properties 

S-strain cells 


fractionation of a cell- 
extract into classes of 

1 1 

1 I 

RNA protein DNA lipid carbohydrate 


molecules tested for transformation of R-strain cells 

• • • • • 

•• ®o m °© •• 

strain strain strain strain strain 

CONCLUSION: The molecule that 
carries the heritable information 
is DNA. 

Figure 5-4 Avery, MacLeod, and McCarty demonstrated 
that DNA is the genetic material. The researchers prepared 
an extract from the disease-causing S strain of pneumococci 
and showed that the "transforming principie" that would 
permanently change the harmless R-strain pneumococci into the 
pathogenic S strain is DNA. This was the first evidence that DNA 
could serve as the genetic material. 

characteristic of DNA; furthermore, they showed that 
enzymes that destroy proteins and RNA did not affect 
the ability of the extract to transform bacteria, while 
enzymes that destroy DNA inactivated it. And like 
Griffith before them, the investigators found that their 
purified preparation changed the bacteria permanently: 
DNA from the pathogenic species was taken up by the 
harmless species, and this change was faithfully passed 
on to subsequent generations of bacteria. 

This landmark study offered rigorous proof that purified 
DNA can act as genetic material. But the resulting paper, 
published in 1944, drew remarkably little attention. 
Despite the meticulous care with which these experi- 
ments were performed, geneticists were not immediately 
convinced that DNA is the hereditary material. Many 
argued that the transformation might have been caused 
by some trace protein contaminant in the preparations. 
Or that the extract might contain a mutagen that alters 
the genetic material of the harmless bacteria—convert- 
ing them to the pathogenic form—rather than containing 
the genetic material itself. 

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CHAPTER 5 DNA and Chromosomes 

Virus cocktails 

The debate was not settled definitively until 1952, when 
Alfred Hershey and Martha Chase fired up their labora- 
tory blender and demonstrated, once and for all, that 
genes are made of DNA. The researchers were study- 
ing T2—a virus that infects and eventually destroys the 
bacterium E. coli. These bacteria-killing viruses behave 
like little molecular syringes: they inject their genetic 
material into the bacterial host cell, while the empty 
virus heads remain attached outside (Figure 5-5A). 
Once inside the bacterial cell, the viral genes direct the 
formation of new virus particles. In less than an hour, 
the infected cells explode, spewing thousands of new 
viruses into the médium. These then infect neighboring 
bacteria, and the process begins again. 

The beauty of T2 is that these viruses contain only two 
kinds of molecules: DNA and protein. So the genetic 
material had to be one or the other. But which? The 
experiment was fairly straightforward. Because the 
viral DNA enters the bacterial cell, while the rest of the 
virus particle remains outside, the researchers decided 
to radioactively label the protein in one batch of virus 
and the DNA in another. Then, all they had to do was 
follow the radioactivity to see whether viral DNA or 

viral protein wound up inside the bacteria. To do this, 
Hershey and Chase incubated their radiolabeled viruses 
with E. coli; after allowing a few minutes for infection to 
take place, they poured the mix into a Waring blender 
and hit "puree." The blender's spinning blades sheared 
the empty virus heads from the surfaces of the bacte¬ 
rial cells. The researchers then centrifuged the sample 
to sepárate the heavier, infected bacteria, which formed 
a pellet at the bottom of the centrifuge tube, from the 
empty viral coats, which remained in suspensión (Figure 

As you have probably guessed, Hershey and Chase 
found that the radioactive DNA entered the bacterial 
cells, while the radioactive proteins remained outside 
with the empty virus heads. They found that the radioac¬ 
tive DNA was also incorporated into the next generation 
of virus particles. 

This experiment demonstrated conclusively that viral 
DNA enters bacterial host cells, whereas viral protein 
does not. Thus, the genetic material in this virus had 
to be made of DNA. Together with the studies done by 
Avery, MacLeod, and McCarty, this evidence clinched 
the case for DNA as the agent of heredity. 



f. coli 

Figure 5-5 Hershey and Chase showed definitively that genes are made of DNA. (A) The researchers worked with T2 viruses, which 
are made entirely of protein and DNA. Each virus acts as a molecular syringe, ¡njecting its genetic material into a bacterium; the empty 
viral capsule remains attached to the outside of the cell. (B) To determine whether the genetic material of the virus is protein or DNA, 
the researchers radioactively labeled the DNA in one batch of viruses with 32 P and the proteins in a second batch of viruses with 35 S. 
Because DNA lacks sulfur and the proteins lack phosphorus, these radioactive ¡sotopes provided a handy way for the researchers to 
distinguish these two types of molecules. These labeled viruses were allowed to infecí and replícate inside E. coli, and the mixture was 
then disrupted by brief pulsing in a Waring blender and separated to part the infected bacteria from the empty viral heads. When the 
researchers measured the radioactivity, they found that much of the 32 P-labeled DNA had entered the bacterial cells, while the vast 
majority of the 35 S-labeled proteins remained in solution with the spent viral particles. 

LibertadDigital \ 2015 

The Structure of DNA 


As we saw in Chapter 2 (Panel 2-6, pp. 76-77), nucleotides are com- 
posed of a nitrogen-containing base and a five-carbon sugar, to which 
is attached one or more phosphate groups. For the nucleotides in DNA, 
the sugar is deoxyribose (henee the ñame deoxyribonucleic acid), and the 
base can be either adenine (A), cytosine (Q, guanine (G), or thymine (T). The 
nucleotides are covalently linked together in a chain through the sugars 
and phosphates, which thus form a backbone of altemating sugar-phos- 
phate-sugar-phosphate (see Figure 5-2B). Because it is only the base that 
differs in each of the four types of subunits, each polynucleotide chain 
in DNA can be thought of as a necklace: a sugar-phosphate backbone 
strung with four types of beads (the four bases A, C, G, and T). These 
same symbols (A, C, G, and T) are also commonly used to denote the 
four different nucleotides—that is, the bases with their attached sugar 

The way in which the nucleotide subunits are linked together gives a 
DNA strand a Chemical polarity. If we imagine that each nucleotide has a 
knob (the phosphate) and a hole (see Figure 5-2A), each strand, formed 
by interlocking knobs with holes, will have all of its subunits lined up 
in the same orientation. Moreover, the two ends of the strand can be 
easily distinguished, as one will have a hole (the 3' hydroxyl) and the 
other a knob (the 5' phosphate). This polarity in a DNA strand is indicated 
by referring to one end as the 3' end and the other as the 5’ end. This 
convention is based on the details of the Chemical linkage between the 
nucleotide subunits. 

The two polynucleotide chains in the DNA double helix are held together 
by hydrogen-bonding between the bases on the different strands. All the 
bases are therefore on the inside of the double helix, with the sugar-phos¬ 
phate backbones on the outside (see Figure 5-2D). The bases do not pair 
at random, however: A always pairs with T, and G always pairs with C 
(Figure 5-6) . In each case, a bulkier two-ring base (a purine, see Panel 2-6, 
pp. 76-77) is paired with a single-ring base (a pyrimidine). Each purine- 
pyrimidine pair is called a base pair, and this complementary base-pairíng 
enables the base pairs to be packed in the energetically most favorable 

Figure 5-6 The two strands of the 
DNA double helix are held together by 
hydrogen bonds between complementary 
base pairs. (A) The shapes and Chemical 
structure of the bases allow hydrogen 
bonds to form efficiently only between A 
and T and between G and C, where atoms 
that are able to form hydrogen bonds (see 
Panel 2-2, pp. 68-69) can be brought cióse 
together without perturblng the double 
helix. Two hydrogen bonds form between 
A and T, whereas three form between 
G and C. The bases can pair in this way 
only if the two polynucleotide chains that 
contain them are antiparallel—that is, 
oriented in opposite directions. (B) A short 
section ofthe double helix viewed from 
its side. Four base pairs are shown. The 
nucleotides are linked together covalently 
by phosphodiester bonds through the 
3'-hydroxyl (-OH) group of one sugar 
and the 5'-phosphate (-OPO3) ofthe 
next (see Panel 2-6, pp. 76-77, to review 
how the carbón atoms in the sugar ring 
are numbered). This linkage gives each 
polynucleotide strand a Chemical polarity; 
that is, its two ends are chemically different. 
The 3' end carries an unlinked -OH group 
attached to the 3' position on the sugar 
ring; the 5' end carries a free phosphate 
group attached to the 5' position on the 
sugar ring. 

LibertadDigital \ 2015 


CHAPTER 5 DNA and Chromosomes 


Which of the following statements 
are corred? Explain your answers. 

A. A DNA strand has a polarity 
because its two ends contain 
different bases. 

B. G-C base pairs are more stable 
than A-T base pairs. 

(A) molecular biology ¡s... 

(0 -- 



Figure 5-8 Linear messages come ¡n 
many forms. The languages shown are 
(A) English, (B) a musical score, (C) Morse 
code, (D) Chínese, and (E) DNA. 

Figure 5-7 A space-filling model shows the conformation of the 
DNA double helix. The two DNA strands wind around each other to 
form a ríght-handed helix (see Figure 4-14) with 10 bases perturn. 
Shown here are 1.5 turns of the DNA double helix. The coiling of the 
two strands around each other creates two grooves in the double 
helix. The wider groove is called the major groove and the smaller 
one the minor groove. The colors of the atoms are: N, blue; O, red; 

P, yellow ; and H, white. 

arrangement in the interior of the double helix. In this arrangement, each 
base pair has a similar width, thus holding the sugar-phosphate back- 
bones an equal distance apart along the DNA molecule. The members 
of each base pair can fit together within the double helix because the 
two strands of the helix run antiparallel to each other—that is, they are 
oriented with opposite polarities (see Figure 5-2C and D). The antiparallel 
sugar-phosphate strands then twist around each other to form a double 
helix containing 10 base pairs per helical turn (Figure 5-7). This twisting 
also contributes to the energetically favorable conformation of the DNA 
double helix. 

A consequence of the base-pairing requirements is that each strand of 
a DNA double helix contains a sequence of nucleotides that is exactly 
complementary to the nucleotide sequence of its partner strand—an A 
always matches a T on the opposite strand, and a C always matches a 
G. This complementarity is of crucial importance when it comes to both 
copying and repairing the DNA, as we discuss in Chapter 6. An animated 
versión of the DNA structure can be seen in Movie 5.1 . 

The Structure of DNA Provides a Mechanism for Heredity 

The need for genes to encode information that must be copied and trans- 
mitted accurately when a cell divides raised two fundamental questions: 
how can the information for specifying an organism be carried in Chemical 
form, and how can the information be accurately copied? The discov- 
ery of the structure of the DNA double helix was a landmark in biology 
because it immediately suggested the answers—and thereby resolved the 
problem of heredity at the molecular level. In this chapter, we outline the 
answer to the first question; in the next chapter, we address in detail the 
answer to the second. 

Information is encoded in the order, or sequence, of the nucleotides along 
each DNA strand. Each base—A, C, T, or G—can be considered a letter in 
a four-letter alphabet that is used to spell out biological messages (Figure 
5-8). Organisms differ frorn one another because their respective DNA 
molecules have different nucleotide sequences and, consequently, carry 
different biological messages. But how is the nucleotide alphabet used to 
make up messages, and what do they spell out? 

It had already been established some time before the structure of DNA 
was determined that genes contain the instructions for producing pro- 
teins. DNA messages, therefore, must somehow be able to encode 
proteins. Consideration of the Chemical character of proteins makes the 
problem easier to define. As discussed in Chapter 4, the function of a pro- 
tein is determined by its three-dimensional structure, and this structure in 
tum is determined by the sequence of the amino acids in its polypeptide 
chain. The linear sequence of nucleotides in a gene must therefore be 
able to spell out the linear sequence of amino acids in a protein. 

The exact correspondence between the 4-letter nucleotide alphabet of 
DNA and the 20-letter amino acid alphabet of proteins—the genetic 
code—is not obvious from the structure of the DNA molecule, and it took 
more than a decade after the discovery of the double helix to work it 

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The Structure of Eukaryotic Chromosomes 


Figure 5-9 Most genes contain ¡nformation to make proteins. As 

we discuss in Chapter 7, each protein-coding gene is used to produce 
RNA molecules, which then direct the production of the specific 
protein molecules. 

out. In Chapter 7, we describe this code in detail when we discuss gene 
expression—the process by which the nucleotide sequence of a gene is 
transcribed into the nucleotide sequence of an RNA molecule, which, in 
most cases, is then translated into the amino acid sequence of a protein 

(Figure 5-9). 

The amount of information in an organism’s DNA is staggering: writ- 
ten out in the four-letter nucleotide alphabet, the nucleotide sequence of 
a very small protein-coding gene frorn humans occupies a quarter of a 
page of text, while the complete human DNA sequence would fill more 
than 1000 books the size of this one. Herein lies a problem that affects the 
architecture of all eukaryotic chromosomes: how can all this information 
be packed neatly into every cell nucleus? In the remainder of this chapter, 
we discuss the answer to this question. 

gene A gene B gene C 



i i r 

^ -NX '■N-* 


I i i 

protein A protein B protein C 


Large amounts of DNA are required to encode all the information needed 
to make even a single-celled bacterium, and far more DNA is needed to 
encode the information to make a multicellular organism like you. Each 
human cell contains about 2 meters (m) of DNA; yet the cell nucleus is 
only 5-8 pm in diameter. Tucking all this material into such a small space 
is the equivalent of trying to fold 40 km (24 miles) of extremely fine thread 
into a tennis ball. 

In eukaryotic cells, very long double-stranded DNA molecules are pack- 
aged into chromosomes. These DNA molecules not only fit readily inside 
the nucleus, but, after they are replicated, they can be easily apportioned 
between the two daughter cells at each cell división. The complex task of 
packaging DNA is accomplished by specialized proteins that bind to and 
fold the DNA, generating a series of coils and loops that provide increas- 
ingly higher levels of organization and prevent the DNA from becoming 
a tangled, unmanageable mess. Amazingly, the DNA is compacted in a 
way that allows it to remain accessible to all of the enzymes and other 
proteins that replícate it, repair it, and control the expression of its genes. 
Bacteria typically carry their genes on a single, circular DNA molecule. 
This molecule is also associated with proteins that condense the DNA, 
but these proteins differ from the ones that package eukaryotic DNA. 
Although this prokaryotic DNA is called a bacterial "chromosome," it 
does not have the same structure as eukaryotic chromosomes, and less is 
known about how it is packaged. Our discussion of chromosome structure 
in this chapter will therefore focus entirely on eukaryotic chromosomes. 

Eukaryotic DNA Is Packaged into Múltiple Chromosomes 

In eukaryotes, such as ourselves, the DNA in the nucleus is distributed 
among a set of different chromosomes. The DNA in a human nucleus, 
for example, contains approximately 3.2 * 10 9 nucleotides parceled out 
into 23 or 24 different types of chromosome (males, with their Y chromo¬ 
some, have an extra type of chromosome that females do not have). Each 
chromosome consists of a single, enormously long, linear DNA molecule 
associated with proteins that fold and pack the fine thread of DNA into 
a more compact structure. The complex of DNA and protein is called 
chromatin. In addition to the proteins involved in packaging the DNA, 

LibertadDigital \ 2015 

CHAPTER 5 DNA and Chromosomes 

Figure 5-10 Each human chromosome 
can be "painted" a different color to 
allow its unambiguous Identification. The 

chromosomes shown here were isolated 
from a cell undergoing nuclear división 
(mitosis) and are therefore in a highly 
compact (condensed) State. Chromosome 
painting ¡s carried out by exposing the 
chromosomes to a collection of human 
DNA molecules that have been coupled 
to a combination of fluorescent dyes. For 
example, DNA molecules derived from 
Chromosome 1 are labeled with one specific 
dye combination, those from Chromosome 
2 with another, and so on. Because the 
labeled DNA can form base pairs (hybridize) 
only to its chromosome of origin (discussed 
in Chapter 10), each chromosome is 
differently colored. For such experiments, 
the chromosomes are treated so that the 
individual strands of the double-helical DNA 
molecules partly sepárate to enable base- 
pairing with the labeled, single-stranded 
DNA, while keeping the chromosome 
structure relatively intact. (A) Micrograph 
shows the array of chromosomes as they 
originally spilled from the lysed cell. 

(B) The same chromosomes have been 
artificially lined up in order. In this so-called 
karyotype, the homologous chromosomes 
are numbered and arranged in pairs; the 
presence of a Y chromosome reveáis that 
these chromosomes carne from a male. 
(From E. Schróck et al., Science 273:494- 
497, 1996. With permission from the AAAS.) 

¿ Y'S> i! » 

1 2 3 4 5 

)| ¡I H iS II >\ 

\ V 6 7 8 9 10 11 12 

chromosomes are also associated with many other proteins involved in 
DNA replication, DNA repair, and gene expression. 

With the exception of the germ cells (sperm and eggs) and highly spe- 
cialized cells that lack DNA entirely (such as mature red blood cells), 
human cells each contain two copies of each chromosome, one inherited 
from the mother and one from the father. The maternal and paternal 
chromosomes of a pair are called homologous chromosomes (homologs ). 
The only nonhomologous chromosome pairs are the sex chromosomes 
in males, where a Y chromosome is inherited from the father and an 
X chromosome from the mother. (Females inherit one X chromosome 
from each parent and have no Y chromosome.) 

In addition to being different sizes, the different human chromosomes 
can be distinguished from one another by a variety of techniques. Each 
chromosome can be "painted" a different color using sets of chromo- 
some-specific DNA molecules coupled to different fluorescent dyes 
(Figure 5-10). This involves a technique called DNA hybridization, which 
takes advantage of complementary base-pairing, as we will describe in 
detail in Chapter 10. A more traditional way of distinguishing one chro¬ 
mosome from another is to stain the chromosomes with dyes that bind to 
certain types of DNA sequences. These dyes mainly distinguish between 
DNA that is rich in A-T nucleotide pairs and DNA that is G-C rich, and 
they produce a predictable pattem of bands along each type of chromo¬ 
some. The pattems that result allow each chromosome to be identified 
and numbered. 

An ordered display of the full set of 46 human chromosomes is called 
the human karyotype (see Figure 5-10). If parts of a chromosome are 
lost, or switched between chromosomes, these changes can be detected. 
Cytogeneticists analyze karyotypes to detect chromosomal abnormalities 
that are associated with some inherited defects (Figure 5-11) and with 
certain types of cáncer. 

Chromosomes Contain Long Strings of Genes 

The most important function of chromosomes is to carry the genes—the 
functional units of heredity (Figure 5-12). A gene is often defined as a 





(A) (B) 

Figure 5-11 Abnormal chromosomes are associated with some 
inherited genetic defects. (A) A pair of Chromosomes 12 from 
a patient with inherited ataxia, a genetic disease of the brain 
characterized by Progressive deterioration of motor skills. The 
patient has one normal Chromosome 12 ( left ) and one abnormally 
long Chromosome 12, which contains a piece of Chromosome 4 as 
identified by its banding pattern. (B) This interpretation was confirmed 
by chromosome painting, in which Chromosome 12 was painted blue 
and Chromosome 4 was painted red. (From E. Schróck et al., Science 
273:494-497, 1996. With permission from the AAAS.) 

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The Structure of Eukaryotic Chromosomes 



0.5% of the DNA of the yeast genome 


10,000 nudeotide pairs genes 

Figure 5-12 Genes are arranged along chromosomes. This figure shows a small región ofthe DNAdouble 
helix in one chromosome from the budding yeast S. cerevisiae. The S. cerevisiae genome contains about 12 
million nudeotide pairs and 6600 genes—spread across 16 chromosomes. Note that, in each gene, only one of 
the two DNA strands actually encodes the information to make an RNA molecule, and this can be either strand, as 
indicated by the Hght red bars. However, a gene is generally denoted to contain both the "coding strand" and its 
complement, as in Figure 5-9. The high density of genes is characteristic of S. cerevisiae. 

segment of DNA that contains the instructions for making a particular 
protein or RNA molecule. Most of the RNA molecules encoded by genes 
are subsequently used to produce a protein (see Figure 5-9). In some 
cases, however, the RNA molecule is the final product; like proteins, 
these RNA molecules have diverse functions in the cell, including struc- 
tural, catalytic, and gene regulatory roles, as we discuss in later chapters. 
Together, the total genetic information carried by all the chromo¬ 
somes in a cell or organism constitutes its genome. Complete genome 
sequences have been determined for thousands of organisms, from 
E. coli to humans. As might be expected, some correlation exists between 
the complexity of an organism and the number of genes in its genome. 
For example, the total number of genes ranges from less than 500 for a 
simple bacterium to about 30,000 for humans. Bacteria and some sin- 
gle-celled eukaryotes, including S. cerevisiae, have especially compact 
genomes: the DNA molecules that make up their chromosomes are little 
more than strings of closely packed genes (see Figure 5-12). However, 
chromosomes from many eukaryotes—including humans—contain, in 
addition to genes and the specific nudeotide sequences required for nor¬ 
mal gene expression, a large excess of interspersed DNA. This extra DNA 
is sometimes called "junk DNA," because the usefulness to the cell has 
not yet been demonstrated. Although the particular nudeotide sequence 
of most of this DNA might not be important, the DNA itself— acting as 
spacer material—may be crucial for the long-term evolution of the spe- 
cies and for the proper activity of the genes. In addition, comparisons of 
the genome sequences from many different species reveal that a portion 
of this extra DNA is highly conserved among related species, indicating 
that it serves an important function—although we don't yet know what 
that is. 

In general, the more complex an organism, the larger is its genome. 
But this relationship does not always hold true. The human genome, for 
example, is 200 times larger than that of the yeast S. cerevisiae, but 30 
times smaller than that of some plants and at least 60 times smaller than 
some species of amoeba (see Figure 1-40). Furthermore, how the DNA is 
apportioned over chromosomes also differs from one species to another. 
Humans have a total of 46 chromosomes (including both maternal and 
paternal sets), but a species of small deer has only 7, while some carp 
species have more than 100. Even closely related species with similar 
genome sizes can have veiy different numbers and sizes of chromo¬ 
somes (Figure 5-13). Thus, although gene number is roughly correlated 
with species complexity, there is no simple relationship between gene 
number, chromosome number, and total genome size. The genomes and 
chromosomes of modern species have each been shaped by a unique his- 
tory of seemingly random genetic events, acted on by specific selection 
pressures, as we discuss in Chapter 9. 

LibertadDigital \ 2015 


CHAPTER 5 DNA and Chromosomes 

Chínese muntjac Indian muntjac 

Figure 5-13 Two dosely related species 
can have similar genome sizes but very 
different chromosome numbers. In the 

evolution of the Indian muntjac deer, 
chromosomes that were ¡nitially sepárate, 
and that remain sepárate ¡n the Chínese 
species, fused without having a major effect 
on the number of genes—or the animal. 
(Courtesy of Deborah Carreno, Natural 
Wonders Photography.) 

Specialized DNA Sequences Are Required for DNA 
Replicaron and Chromosome Segregation 

To form a functional chromosome, a DNA molecule musí do more than 
simply carry genes: it musí be able to be replicated, and the replicated 
copies must be separated and partitioned equally and reliably into the 
two daughter cells at each cell división. These processes occur through 
an ordered series of events, known collectively as the cell cycle. This 
cycle of cell growth and división is briefly summarized in Figure 5-14 and 
will be discussed in detail in Chapter 18. Only two broad stages of the cell 
cycle need concern us in this chapter: interphase, when chromosomes 
are duplicated, and mitosis, when they are distributed, or segregated, to 
the two daughter nuclei. 

During interphase, the chromosomes are extended as long, thin, tan- 
gled threads of DNA in the nucleus and cannot be easily distinguished 
in the light microscope (see Figure 5-1). We refer to chromosomes in 
this extended State as interphase chromosomes. As we discuss in Chapter 
6, specialized DNA sequences found in all eukaryotes ensure that DNA 
replication occurs efficiently during interphase. One type of nucleotide 
sequence acts as a replication origin, where replication of the DNA 
begins; eukaryotic chromosomes contain many replication origins to 
ensure that the long DNA molecules are replicated rapidly (Figure 5-15). 
Another DNA sequence forms the telomeres at each of the two ends of a 
chromosome. Telomeres contain repeated nucleotide sequences that are 
required for the ends of chromosomes to be replicated. They also cap the 
ends of the DNA molecule, preventing them from being mistaken by the 
cell as broken DNA in need of repair. 

nuclear envelope 









Figure 5-14 The duplication and segregation of chromosomes occurs through an ordered cell cycle in proliferating cells. During 
interphase, the cell expresses many of its genes, and—during part of this phase—it duplicates chromosomes. Once chromosome 
duplication is complete, the cell can enter M ph ase, during which nuclear división, or mitosis, occurs. In mitosis, the duplicated 
chromosomes condense, gene expression largely ceases, the nuclear envelope breaks down, and the mitotic spindle forms from 
microtubules and other proteins. The condensed chromosomes are then captured by the mitotic spindle, one complete set is pulled 
to each end of the cell, and a nuclear envelope forms around each chromosome set. In the final step of M phase, the cell divides to 
produce two daughter cells. Only two different chromosomes are shown here for simplicity. 

LibertadDigital \ 2015 

The Structure of Eukaryotic Chromosomes 


telomere — 

portion of 
mitotic spindle duplicated 

in sepárate cells 

Figure 5-15 Three DNA sequence 
elements are needed to produce a 
eucaryotic chromosome that can be 
replicated and then segregated at 
mitosis. Each chromosome has múltiple 
origins of replication, one centromere, and 
two telomeres. The sequence of events 
that a typical chromosome follows durlng 
the cell cycle is shown schematically. The 
DNA replicates in interphase, beginning at 
the origins of replication and proceeding 
bidirectionally from the origins across the 
chromosome. In M phase, the centromere 
attaches the duplicated chromosomes 
to the mitotic spindle so that one copy is 
distributed to each daughter cell when 
the cell divides. Prior to cell división, the 
centromere also helps to hold the compact, 
duplicated chromosomes together 
until they are ready to be pulled apart. 
Telomeres, which form special caps at 
the tips of each chromosome, aid in the 
replication of chromosome ends. 

Eukaryotic chromosomes also contain a third type of specialized DNA 
sequence, called the centromere, that allows duplicated chromosomes 
to be separated during M phase (see Figure 5-15). During this stage of 
the cell cycle, the DNA coils up, adopting a more and more compact 
structure, ultimately forming highly compacted, or condensed, mitotic 
chromosomes. This is the State in which the duplicated chromosomes can 
be most easily visualized (Figure 5-16 and see Figures 5-1 and 5-14). 
Once the chromosomes have condensed, the centromere attaches the 
mitotic spindle to each duplicated chromosome in a way that allows one 
copy of each chromosome to be segregated to each daughter cell (see 
Figure 5-15B). We describe the central role that centromeres play in cell 
división in Chapter 18. 

Interphase Chromosomes Are Not Randomly Distributed 
Within the Nucleus 

Inside the nucleus, the interphase chromosomes—although longer and 
finer than mitotic chromosomes—are nonetheless organized in various 





Figure 5-16 A typical duplicated mitotic 
chromosome is highly compact. Because 
DNA is replicated during interphase, each 
duplicated mitotic chromosome contains 
two identical daughter DNA molecules (see 
Figure 5-15A). Each of these very long 
DNA molecules, with its associated 
proteins, is called a chromatid ; once 
the two sister chromatids sepárate, they 
are considered individual chromosomes. 

(A) Ascanning electrón micrograph of a 
mitotic chromosome. The two chromatids 
are tightly joined together. The constricted 
región reveáis the position of the 
centromere. (B) A cartoon representaron 
of a mitotic chromosome. (A, courtesy of 
Terry D. Alien.) 

LibertadDigital \ 2015 


CHAPTER 5 DNA and Chromosomes 

Figure 5-17 Interphase chromosomes 
occupy their own distinct territories 
within the nucleus. DNA probes coupled 
with different fluorescent markers were used 
to paint individual interphase chromosomes 
in a human cell. Viewed ¡n a fluorescence 
microscope, each interphase chromosome 
¡s seen to occupy its own discrete territory 
within the nucleus, ratherthan being mixed 
with the other chromosomes like spaghetti 
in a bowl. Note that pairs of homologous 
chromosomes, such as the two copies of 
Chromosome 9 indicated, are not generally 
located in the same position. (From 
M.R. Speicher and N.P. Cárter, Nat. Rev. 
Genet. 6:782-792, 2005. With permission 
from Macmillan Publishers Ltd.) 

ways. First, each chromosome tends to occupy a particular región of 
the interphase nucleus, and so different chromosomes do not become 
extensively entangled with one another (Figure 5-17). In addition, some 
chromosomes are attached to particular sites on the nuclear envelope— 
the pair of concentric membranes that surround the nucleus—or to the 
underlying nuclear lamina, the protein meshwork that supports the enve¬ 
lope (discussed in Chapter 17). 

The most obvious example of chromosome organization in the inter¬ 
phase nucleus is the nucleolus (Figure 5-18). The nucleolus is where 
the parts of the different chromosomes carrying genes that encode ribos- 
omal RNAs cluster together. Here, ribosomal RNAs are synthesized and 
combine with proteins to form ribosomes, the cell’s protein-synthesizing 
machines. As we discuss in Chapter 7, ribosomal RNAs play both struc- 
tural and catalytic roles in the ribosome. 

The DNA in Chromosomes Is Always Highly Condensed 

As we have seen, all eukaryotic cells, whether in interphase or mito- 
sis, package their DNA tightly into chromosomes. Human Chromosome 
22, for example, contains about 48 million nucleotide pairs; stretched 
out end-to-end, its DNA would extend about 1.5 cm. Yet, during mito- 
sis, Chromosome 22 measures only about 2 pm in length—that is, nearly 
10,000 times more compact than the DNA would be if it were stretched 
to its full length. This remarkable feat of compression is performed by 
proteins that coil and fold the DNA into higher and higher levels of organ¬ 
ization. The DNA of interphase chromosomes, although about 20 times 
less condensed than that of mitotic chromosomes (Figure 5-19), is still 
packed tightly. 

Figure 5-18 The nucleolus is the most 
prominent structure in the interphase 
nucleus. Electron mlcrograph of a thin 
section through the nucleus of a human 
fibroblast. The nucleus ¡s surrounded by the 
nuclear envelope. Inside the nucleus, the 
chromatin appears as a diffuse speckled 
mass, with regions that are especially dense, 
called heterochromatln (dark staining). 
Heterochromatin contains few genes and 
is located mainly around the perlphery of 
the nucleus, immediately underthe nuclear 
envelope. The large dark región is the 
nucleolus, which contains the genes for 
ribosomal RNAs; these genes are located 
on múltiple chromosomes but are clustered 
together in the nucleolus. (Courtesy of 
E.G. Jordán and J. McGovern.) 

LibertadDigital \ 2015 

The Structure of Eukaryotic Chromosomes 


In the next sections, we introduce the specialized proteins that make this 
compression possible. Bear in mind, though, that chromosome structure 
is dynamic. Not only do chromosomes condense and decondense dur- 
ing the cell cycle, but chromosome packaging must be flexible enough 
to allow rapid, on-demand access to different regions of the interphase 
chromosome, unpacking enough to allow protein complexes access 
to specific, localized DNA sequences for replication, repair, or gene 

Nucleosomes Are the Basic Units of Eukaryotic 
Chromosome Structure 

The proteins that bind to DNA to form eukaryotic chromosomes are tradi- 
tionally divided into two general classes: the histones and the nonhistone 
chromosomalproteins. Histones are present in enormous quantities (more 
than 60 million molecules of several different types in each cell), and their 
total mass in chromosomes is about equal to that of the DNA itself. The 
complex of both classes of protein with nuclear DNA is called chromatin. 
Histones are responsible for the first and most fundamental level of chro¬ 
matin packing, the nucleosome, which was discovered in 1974. When 
interphase nuclei are broken open very gently and their contents exam- 
ined with an electrón microscope, much of the chromatin is in the form 
of chromatin Jibers with a diameter of about 30 nm (Figure 5-20A). If this 
chromatin is subjected to treatments that cause it to unfold partially, it 
can then be seen in the electrón microscope as a series of "beads on a 
string" (Figure 5-20B). The string is DNA, and each bead is a nucleosome 
core partióle, which consists of DNA wound around a core of proteins 
formed from histones. 

The structure of the nucleosome core particle was determined after first 
isolating nucleosomes by treating chromatin in its unfolded, "beads on a 
string" form with enzymes called nucleases, which break down DNA by 
cutting the phosphodiester bonds between núcleotides. After digestión 
for a short period, only the exposed DNA between the core particles— 
the linker DNA— is degraded, allowing the core particles to be isolated. 
An individual nucleosome core particle consists of a complex of eight 
histone proteins—two molecules each of histones H2A, H2B, H3, and 
H4—and a stretch of double-stranded DNA, 147 nucleotide pairs long, 
that winds around this histone octamer (Figure 5-21 ). The high-resolution 
structure of the nucleosome core particle was solved in 1997, revealing 
in atomic detail the disc-shaped histone octamer around which the DNA 
is tightly wrapped, making 1.7 turns in a left-handed coil (Figure 5-22). 

Figure 5-19 DNA in interphase 
chromosomes ¡s less compact than ¡n 
mitotic chromosomes. (A) An electrón 
micrograph showing an enormous tangle of 
chromatin (DNA with its associated proteins) 
spilling out of a lysed interphase nucleus. 

(B) Schematic drawing of a human mitotic 
chromosome drawn to the same scale. 
(Courtesy of Victoria Foe.) 

Figure 5-20 Nucleosomes can be seen in 
the electrón microscope. (A) Chromatin 
isolated directly from an interphase nucleus 
appears in the electrón microscope as 
a chromatin fiber about 30-nm thick; 
a part of one such fiber ¡s shown here. 

(B) This electrón micrograph shows a 
length of a chromatin fiber that has been 
experlmentally unpacked, or decondensed, 
after isolation to show the "beads-on-a- 
string" appearance of the nucleosomes. 

(A, courtesy of Barbara Hamkalo; 

B, courtesy of Victoria Foe.) 

LibertadDigital \ 2015 

CHAPTER 5 DNA and Chromosomes 

core histories 

linker DNA of nucleosome 


"beads-on-a-string" nucleosome ineludes 
form of chromatin ~200 nucleotide 

pairs of DNA 


core partide 



histone 147-nucleotide-pair 

octamer DNA double helix 


í - '1 ¡ l 

•* *# 

Figure 5-21 Nucleosomes contain DNA wrapped around a protein 
core of eight histone molecules. In a test tube, the nucleosome core 
particle can be released from chromatin by digestión of the linker DNA 
with a nuclease, which degrades the exposed DNA but not the DNA 
wound tightly around the nucleosome core. The DNA around each 
¡solated nucleosome core particle can then be released and its length 
determined. With 147 nucleotide pairs in each fragment, the DNA 
wraps almosttwice around each histone octamer. 

The linker DNA between each nucleosome core particle can vary in 
length from a few nucleotide pairs up to about 80. (The term nucleosome 
technically refers to a nucleosome core particle plus one of its adjacent 
DNA linkers, as shown in Figure 5-21, but it is often used to refer to the 
nucleosome core particle itself.) The formation of nucleosomes converts 
a DNA molecule into a chromatin thread that is approximately one-third 
the length of the initial piece of DNA, and it provides the first level of DNA 

All four of the histones that make up the octamer are relatively small 
proteins, with a high proportion of positively charged amino acids (lysine 
and arginine). The positive charges help the histones bind tightly to the 
negatively charged sugar-phosphate backbone of DNA. These numer- 
ous electrostatic interactions explain in part why DNA of virtually any 
sequence can bind to a histone octamer. Each of the histones in the 

Q histone H2A Q histone H2B Q histone H3 Q histone H4 

Figure 5-22 The structure of the nucleosome core particle, as determined by 
X-ray diffraction analysis, reveáis how DNA is tightly wrapped around a 
disc-shaped histone octamer. Two views of a nucleosome core particle are shown 
here. The two strands of the DNA double helix are shown ¡n gray. A portlon of an 
H3 histone tail (green) can be seen extendlng from the nucleosome core particle, 
but the tails of the other histones have been truncated. (Reprinted by permisslon 
from K. Luger et al., Nature 389:251-260, 1997. With permisslon from Macmlllan 
Publlshers Ltd.) 

LibertadDigital \ 2015 

The Structure of Eukaryotic Chromosomes 


octamer also has a long, unstructured N-terminal amino acid "tail" that 
extends out from the nucleosome core particle (see Figure 5-22). These 
histone tails are subject to several types of reversible, covalent Chemical 
modifications that control many aspects of chromatin structure. 

The histones that form the nucleosome core are among the most highly 
conserved of all known eukaryotic proteins: there are only two differ- 
ences between the amino acid sequences of histone H4 from peas and 
cows, for example. This extreme evolutionary conservation reflects the 
vital role of histones in controlling eukaryotic chromosome structure. 

Chromosome Packing Occurs on Múltiple Levels 

Although long strings of nucleosomes form on most chromosomal DNA, 
chromatin in the living cell rarely adopts the extended beads-on-a-string 
form seen in Figure 5-20B. Instead, the nucleosomes are further packed 
on top of one another to generate a more compact structure, such as the 
chromatin fiber shown in Figure 5-20A and Movie 5.2. This additional 
packing of nucleosomes into a chromatin fiber depends on a fifth his¬ 
tone called histone Hl, which is thought to pulí adjacent nucleosomes 
together into a regular repeating array. This "linker" histone changes the 
path the DNA takes as it exits the nucleosome core, allowing it to form a 
more condensed chromatin fiber (Figure 5-23). 

We saw earlier that during mitosis chromatin becomes so highly con¬ 
densed that individual chromosomes can be seen in the light microscope. 
How is a chromatin fiber folded to produce mitotic chromosomes? The 
answer is not yet known in detail, but it is known that the chromatin fiber 
is folded into a series of loops, and that these loops are further condensed 
to produce the interphase chromosome; finally, this compact string of 
loops is thought to undergo at least one more level of packing to form the 
mitotic chromosome (Figure 5-24 and Figure 5-25). 

short región of 
DNA double helix 

form of chromatin 

chromatin fiber 
of packed 

chromatin fiber 
folded into loops 







'00 i 





Figure 5-23 A linker histone helps to pulí 
nucleosomes together and pack them 
into a more compact chromatin fiber. 

Histone Hl consists of a globular región 
plus a pair of long tails at its C-terminal 
and N-terminal ends. The globular región 
constrains an additional 20 base pairs of the 
DNA where it exits from the nucleosome 
core, an activity that is thought to be 
important for the formation of the chromatin 
fiber. The long C-terminal tail is required for 
Hl to bind to chromatin. The positions of 
the C-terminal and N-terminal tails in the 
nucleosome are not known. 


Assuming that the histone 
octamer (shown in Figure 5-21) 
forms a cylinder 9 nm in diameter 
and 5 nm in height and that the 
human genome forms 32 million 
nucleosomes, what volume of 
the nucleus (6 pm in diameter) is 
occupied by histone octamers? 
(Volume of a cylinder is n^h; volume 
of a sphere is 4/3 ítr 3 .) What fraction 
of the total volume of the nucleus 
do the histone octamers occupy? 
How does this compare with the 
volume of the nucleus occupied by 
human DNA? 

Figure 5-24 DNA packing occurs on 
several levels in chromosomes. This 
schematic drawing shows some of the levels 
thought to give rise to the highly condensed 
mitotic chromosome. The actual structures 
are still uncertain. 

LibertadDigital \ 2015 

CHAPTER 5 DNA and Chromosomes 



Histone proteins are among the 
most highly conserved proteins in 
eukaryotes. Histone H4 proteins 
from a pea and a cow, for example, 
differ in only 2 of 102 amino acids. 
Comparison of the gene sequences 
shows many more differences, but 
only two change the amino acid 
sequence. These observations 
indícate that mutations that change 
amino acids must have been 
selected against during evolution. 
Why do you suppose that amino- 
acid-altering mutations in histone 
genes are deleterious? 

Figure 5-25 The mitotic chromosome contains chromatin that 
¡s packed especially tightly. This scanning electrón micrograph 
shows a región near one end of a typical mitotic chromosome. Each 
knoblike projection ¡s believed to represent the tip of a sepárate loop 
of chromatin. The chromosome has duplicated, forming two sister 
chromatids that are still held cióse together (see Figure 5-16). The 
ends of the two chromatids can be distinguished on the right of the 
photo. (From M.P. Marsden and U.K. Laemmli, Ce//17:849-858, 1989. 
With permission from Elsevier.) 


So far, we have discussed how DNA is packed tightly into chromatin. We 
now tum to the question of how this packaging can be regulated to allow 
rapid access to the underlying DNA. The DNA in cells carries enormous 
amounts of coded information, and cells must be able to get to this infor- 
mation as needed. 

In this section, we discuss how a cell can alter its chromatin structure 
to expose localized regions of DNA and allow access to speciflc proteins 
and protein complexes, particularly those involved in gene expression 
and in DNA replication and repair. We then discuss how chromatin struc¬ 
ture is established and maintained—and how a cell can pass on some 
forms of this structure to its descendants. The regulation and inheritance 
of chromatin structure play crucial parts in the development of eukaryo- 
tic organisms. 

Changes in Nucleosome Structure Allow Access to DNA 

Eukaryotic cells have several ways to adjust the local structure of their 
chromatin rapidly. One way takes advantage of chromatin-remodeling 
complexes, protein machines that use the energy of ATP hydrolysis to 
change the position of the DNA wrapped around nucleosomes (Figure 
5-26A). The complexes, which attach to both the histone octamer and 
the DNA wrapped around it, can locally alter the arrangement of nucle¬ 
osomes on the DNA, making the DNA either more accessible (Figure 
5-26B) or less accessible to other proteins in the cell. During mitosis, 
many of the chromatin-remodeling complexes are inactivated, which 
may help mitotic chromosomes maintain their tightly packed structure. 
Another way of altering chromatin structure relies on the reversible 
Chemical modification of the histones. The tails of all four of the core 
histones are particularly subject to these covalent modifications (Figure 
5-27A). For example, acetyl, phosphate, or methyl groups can be added 
to and removed from the tails by enzymes that reside in the nucleus 
(Figure 5-27B). These and other modifications can have important con- 
sequences for the stability of the chromatin fiber. Acetylation of lysines, 
for instance, can reduce the affinity of the tails for adjacent nucleosomes, 
thereby loosening chromatin structure and allowing access to particular 
nuclear proteins. 

Most importantly, however, these modifications can serve as docking 
sites on the histone tails for a variety of regulatory proteins. Different 
pattems of modifications attract different proteins to particular stretches 
of chromatin. Some of these proteins promote chromatin condensation, 
whereas others decondense chromatin and facilítate access to the DNA. 
Speciflc combinations of tail modifications and the proteins that bind to 
them have different meanings for the cell: one pattem, for example, indi- 
cates that a particular stretch of chromatin has been newly replicated; 

LibertadDigital \ 2015 

The Regulation of Chromosome Structure 

Figure 5-26 Chromatin-remodeling 
complexes locally reposition the DNA 
wrapped around nudeosomes. (A) The 

complexes use energy derived from ATP 
hydrolysis to loosen the nucleosomal DNA 
and push ¡t along the histone octamer, 
thereby exposing the DNA to other DNA- 
binding proteins. The blue stripes have 
been added to show how the nucleosome 
moves along the DNA. Many cycles of 
ATP hydrolysis are required to produce 
such a shlft. (B) In the case shown, the 
reposltionlng of nudeosomes decondenses 
the chromatln ¡n a particular chromosomal 
región; ¡n other cases, it condenses the 




another indicates that the genes in that stretch of chromatin should be 
expressed; still others indícate that the nearby genes should be silenced 

(Figure 5-27C). 

Like the chromatin-remodeling complexes, the enzymes that modify 
histone tails are tightly regulated. They are brought to particular chro¬ 
matin regions mainly by interactions with proteins that bind to specific 


gene silencing 

gene expression 

gene expression 

Figure 5-27 The pattern of modification of histone tails can díctate how a stretch of chromatin is treated by the cell. 

(A) Schematlc drawlng showing the posltions of the histone tails that extend from each nucleosome. (B) Each histone can be modified 
by the covalent attachment of a number of different Chemical groups, mainly to the tails. Histone H3, for example, can receive an acetyl 
group (Ac), a methyl group (M), or a phosphate group (P). The numbers denote the positions of the modified amino acids in the protein 
chain, with each amino acid designated by its one-letter code. Note that some positions, such as lysines (K) 9, 14, 23, and 27, can be 
modified in more than one way. Moreover, lysines can be modified with either one, two, or three methyl groups (not shown). Note that 
histone H3 contains 135 amino acids, most of which are in its globular portion ( green ), and that most modifications are on its N-terminal 
tail ( orange). (C) Different combinations of histone tail modifications can confer a specific meaning on the stretch of chromatin on which 
they occur, as indicated. Only a few of these "meanings" are known. 

LibertadDigital \ 2015 


CHAPTER 5 DNA and Chromosomes 

sequences in DNA (we discuss these proteins in Chapter 8). The histone- 
modifying enzymes work in concert with the chromatin-remodeling 
complexes to condense or decondense stretches of chromatin, allowing 
local chromatin structure to change rapidly according to the needs of the 

Interphase Chromosomes Contain Both Condensed and 
More Extended Forms of Chromatin 

The localized alteration of chromatin packing by remodeling complexes 
and histone modification has important effects on the large-scale struc¬ 
ture of interphase chromosomes. Interphase chromatin is not uniformly 
packed. Instead, regions of the chromosome that contain genes that are 
being expressed are generally more extended, while those that contain 
silent genes are more condensed. Thus, the detailed structure of an inter¬ 
phase chromosome can differ frorn one cell type to the next, helping to 
determine which genes are expressed. Most cell types express about 20 
to 30 % of the genes they contain. 

The most highly condensed form of interphase chromatin is called hete- 
rochromatin (from the Greek he teros, "different," plus chromatin). It was 
first observed in the light microscope in the 1930s as discrete, strongly 
staining regions within the mass of chromatin. Heterochromatin typically 
makes up about 10% of an interphase chromosome, and in mammalian 
chromosomes, it is concentrated around the centromere región and in 
the telomeres at the ends of the chromosomes (see Figure 5-15). 

The rest of the interphase chromatin is called euchromatin (from the 
Greek eu, "trae" or "normal," plus chromatin). Although we use the term 
euchromatin to refer to chromatin that exists in a more decondensed 
State than heterochromatin, it is now clear that both euchromatin and 
heterochromatin are composed of mixtures of different chromatin struc- 
tures (Figure 5-28). 

Each type of chromatin structure is established and maintained by 
different sets of histone tail modifications that attract distinct sets of non- 
histone proteins. The modifications that direct the formation of the most 
common type of heterochromatin, for example, inelude the methylation 
of lysine 9 in histone H3 (see Figure 5-27). Once it has been established, 
heterochromatin can spread because these histone tail modifications 
attract a set of heterochromatin-specific proteins, including histone-mod- 
ifying enzymes, which then create the same histone tail modifications on 
adjacent nucleosomes. These modifications in turn recruit more of the 
heterochromatin-specific proteins, causing a wave of condensed chro¬ 
matin to propágate along the chromosome. This heterochromatin will 
continué to spread until it encounters a barrier DNA sequence that stops 
the propagation (Figure 5-29). In this manner, extended regions of het¬ 
erochromatin can be established along the DNA. 

Figure 5-28 The structure of chromatin varíes along a single interphase chromosome. As schematically indicated by different 
colors (and the path of the DNA molecule represented by the central black Une), heterochromatin and euchromatin each represent 
a set of different chromatin structures with different degrees of condensaron. Overall, heterochromatin is more condensed than 

LibertadDigital \ 2015 

The Regulation of Chromosome Structure 


histone tail modifications 

O O O QJT‘0 O O 




o o 

Figure 5-29 Heterochromatin-specific 
modifications allow heterochromatin to 
form and to spread. These modifications 
attract heterochromatin-specific proteins 
that reproduce the same modifications 
on neighboring histones. In this manner, 
heterochromatin can spread until it 
encounters a barrier DNA sequence that 
blocks its propagation into regions of 

Most DNA that is permanently folded into heterochromatin in the cell 
does not contain genes. Because heterochromatin is so compact, genes 
that accidentally become packaged into heterochromatin usually fail to 
be expressed. Such inappropriate packaging of genes in heterochromatin 
can cause disease: in humans, the gene that encodes p-globin—which 
forms part of the oxygen-carrying hemoglobin molecule—is situated next 
to a región of heterochromatin. If, because of an inherited DNA deletion, 
that heterochromatin spreads, the p-globin gene is poorly expressed and 
the person develops a severe form of anemia. 

Perhaps the most striking example of the use of heterochromatin to keep 
genes shut down, or silenced, is found in the interphase X chromosomes 
of female mammals. In mammals, female cells contain two X chromo¬ 
somes, whereas male cells contain one X and one Y. Because a double 
dose of X-chromosome products would be lethal, female mammals have 
evolved a mechanism for permanently inactivating one of the two X 
chromosomes in each cell. At random, one or other of the two X chro¬ 
mosomes in each cell becomes highly condensed into heterochromatin 
early in embryonic development. Thereafter, the condensed and inactive 
State of that X chromosome is inherited in all of the many descendants of 
those cells (Figure 5-30). 

When a cell divides, it generally passes on its histone modifications, chro- 
matin structure, and gene expression pattems to the two daughter cells. 
Such "cell memory" is critical for the establishment and maintenance 
of different cell types during the development of a complex multicellu- 
lar organism. We discuss the mechanisms involved in cell memory in 
Chapter 8, where we consider the control of gene expression. 


Mutations in a particular gene on 
the X chromosome result in color 
blindness in men. By contrast, most 
women carrying the mutation have 
proper color visión but see colored 
objects with reduced resolution, as 
though functional cone cells (the 
photoreceptor cells responsible for 
color visión) are spaced farther apart 
than normal in the retina. Can you 
give a plausible explanation for this 
observation? If a woman is color- 
blind, what could you say about her 
father? About her mother? Explain 
your answers. 

LibertadDigital \ 2015 


CHAPTER 5 DNA and Chromosomes 

Figure 5-30 One of the two X chromosomes 
is inactivated in the cells of mammalian 
females by heterochromatin formation. Each 
female ceII contains two X chromosomes, one 
from the mother (X m ) and the other from the 
father (X p ). At an early stage in embryonic 
development, one of these two chromosomes 
becomes condensed ¡nto heterochromatin 
¡n each cell, apparently at random. At each 
cell división, the same X chromosome 
becomes condensed (and inactivated) in all 
the descendants of that original cell. Thus, 
all mammalian females end up as mixtures 
(mosaics) of cells bearing maternal or paternal 
inactivated X chromosomes. In most of their 
tissues and organs, about half the cells will be of 
one type, and the other half will be of the other. 

only X m active in this clone 

only X p active ¡n 


• Life depends on the stable storage and inheritance of genetic 

• Genetic information is carried by veiy long DNA molecules and is 
encoded in the linear sequence of four nucleotides: A, T, G, and C. 

• Each molecule of DNA is a double helix composed of a pair of 
antiparallel, complementaiy DNA strands, which are held together 
by hydrogen bonds between G-C and A-T base pairs. 

• The genetic material of a eukaryotic cell is contained in a set of chro¬ 
mosomes, each formed from a single, enormously long DNA molecule 
that contains many genes. 

• When a gene is expressed, part of its nucleotide sequence is tran- 
scribed into RNA molecules, many of which are translated into 

• The DNA that forms each eukaryotic chromosome contains, in addi- 
tion to genes, many replication origins, one centromere, and two 
telomeres. These special DNA sequences ensure that, before cell 
división, each chromosome can be duplicated efficiently, and that 
the resulting daughter chromosomes are parceled out equally to the 
two daughter cells. 

• In eukaryotic chromosomes, the DNA is tightly folded by binding to 
a set of histone and nonhistone proteins. This complex of DNA and 
protein is called chromatin. 

• Histones pack the DNA into a repeating array of DNA-protein par- 
ticles called nucleosomes, which further fold up into even more 
compact chromatin structures. 

LibertadDigital \ 2015 

Chapter 5 End-of-Chapter Questions 


A cell can regúlate its chromatin structure—temporarily decondens- 
ing or condensing particular regions of its chromosomes—using 
chromatin-remodeling complexes and enzymes that covalently mod- 
ify histone tails in various ways. 

The loosening of chromatin to a more decondensed State allows pro- 
teins involved in gene expression, DNA replication, and DNA repair to 
gain access to the necessaiy DNA sequences. 

Some forms of chromatin have a pattem of histone tail modification 
that causes the DNA to become so highly condensed that its genes 
cannot be expressed to produce RNA; such condensation occurs on 
all chromosomes during mitosis and in the heterochromatin of inter- 
phase chromosomes. 


base pair 

gene expression 

cell cycle 

genetic code 





chromatin-remodeling complex 






deoxyribonucleic acid (DNA) 


double helix 

replication origin 


telomere gene 




A. The nucleotide sequence of one DNA strand of a DNA 
double helix is 


What is the sequence of the complementary strand? 

B. In the DNA of certain bacterial cells, 13% of the 
nucleotides are adenine. What are the percentages of the 
other nucleotides? 

C. How many possible nucleotide sequences are there for a 
stretch of DNA that is N nucleotides long, if it is (a) single- 
stranded or (b) double-stranded? 

D. Suppose you had a method of cutting DNA at specific 
sequences of nucleotides. How many nucleotides long 
(on average) would such a sequence have to be in order 
to make just one cut in a bacterial genome of 3 x 10 6 
nucleotide pairs? How would the answer differ for the 
genome of an animal cell that contains 3 x 10 9 nucleotide 


An A-T base pair is stabilized by only two hydrogen bonds. 
Hydrogen-bonding schemes of very similar strengths can 
also be drawn between other base combinations that 
normally do not occur in DNA molecules, such as the A-C 
and the A-G pairs shown in Figure Q5-6. 

3' 5' 3' 5' 

Figure Q5-6 

LibertadDigital \ 2015 

194 CHAPTER 5 DNA and Chromosomes 

What would happen ¡f these pairs formed during DNA 
replication and the ¡nappropriate bases were incorporated? 
Discuss why this does not often happen. (Hint: see 
Figure 5-6.) 


A. A macromolecule isolated from an extraterrestrial source 
superficially resembles DNA, but closer analysis reveáis that 
the bases have quite different structures (Figure Q5-7). 
Bases V, W, X, and Y have replaced bases A, T, G, and C. 
Look at these structures closely. Could these DNA-like 
molecules have been derived from a living organism that 
uses principies of genetic inheritance similar to those used 
by organisms on Earth? 

Figure Q5-7 

B. Simply judged by their potential for hydrogen-bonding, 
could any of these extraterrestrial bases replace terrestrial 
A, T, G, or C in terrestrial DNA? Explain your answer. 


The two strands of a DNA double helix can be separated 
by heating. If you raised the temperature of a solution 
containing the following three DNA molecules, in what 
order do you suppose they would "melt"? Explain your 


B. 5'-attataaaatatttagatactatatttacaa-3' 


C. 5'-agagctagatcgat-3' 



The total length of DNA in the human genome is about 
1 m, and the diameter of the double helix is about 2 nm. 
Nucleotides in a DNA double helix are stacked (see 

Figure 5-6B) at an interval of 0.34 nm. If the DNA were 
enlarged so that its diameter equaled that of an electrical 
extensión cord (5 mm), how long would the extensión cord 
be from one end to the other (assuming that it is completely 
stretched out)? How cióse would the bases be to each 
other? How long would a gene of 1000 nucleotide pairs be? 


A compact disc (CD) stores about 4.8 x 10 9 bits of 
information in a 96 cm 2 area. This information is stored as a 
binary code—that is, every bit is either a 0 or a 1. 

A. How many bits would it take to specify each nucleotide 
pair in a DNA sequence? 

B. How many CDs would it take to store the information 
contained in the human genome? 


Which of the following statements are corred? Explain your 

A. Each eukaryotic chromosome must contain the following 
DNA sequence elements: múltiple origins of replication, two 
telomeres, and one centromere. 

B. Nucleosome core partióles are 30 nm in diameter. 


Define the following terms and their relationships to one 

A. Interphase chromosome 

B. Mitotic chromosome 

C. Chromatin 

D. Heterochromatin 

E. Histones 

F. Nucleosome 


Carefully consider the result shown in Figure Q5-13. 

Each of the two colonies shown on the left is a clump of 
approximately 100,000 yeast cells that has grown up from 
a single cell, which is now somewhere in the middle of the 
colony. The two yeast colonies are genetically different, as 
shown by the chromosomal maps on the right. 

white colony of 
yeast cells 

red colony of 
yeast cells 
wlth white sectors 

Figure Q5-13 

LibertadDigital \ 2015 

Chapter 5 End-of-Chapter Questions 


The yeast Ade2 gene encodes one of the enzymes required 
for adenine biosynthesis, and the absence of the Ade2 gene 
product leads to the accumulation of a red pigment. At ¡ts 
normal chromosome location, Ade2 is expressed ¡n all cells. 
When ¡t ¡s positioned near the telomere, which ¡s highly 
condensed, Ade2 ¡s no longer expressed. How do you think 
the white sectors arise? What can you conclude about the 
propagation of the transcriptional State of the Ade2 gene 
from mother to daughter cells? 


The two electrón micrographs in Figure Q5-14 show nuclei 
of two different cell types. Can you tell from these pictures 
which of the two cells is transcribing more of its genes? 
Explain how you arrived at your answer. (Micrographs 
courtesy of Don W. Fawcett.) 


Figure Q5-14 


DNA forms a right-handed helix. Pick out the right-handed 
helix from those shown in Figure Q5-15. 

(A) (B) (C) 

Figure Q5-15 


A single nucleosome core particle is 11 nm in diameter and 
contains 147 bp of DNA (the DNA double helix measures 
0.34 nm/bp). What packing ratio (ratio of DNA length to 
nucleosome diameter) has been achieved by wrapping DNA 
around the histone octamer? Assuming that there are an 
additional 54 bp of extended DNA in the linker between 
nucleosomes, how condensed is "beads-on-a-string" DNA 
relative to fully extended DNA? What fraction of the 
10,000-fold condensation that occurs at mitosis does this 
first level of packing represent? 

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DNA Replication, Repair, and 

The ability of a cell to survive and proliferate in a chaotic environment DNA REPLICATION 

depends on the accurate duplication of the vast quantity of genetic infor- 

mation carried in its DNA. This duplication process, called DNA replication , 

musí occur before a cell can divide to produce two genetically identical DNA KtrAIK 

daughter cells. Maintaining order in a cell also requires the continual 

surveillance and repair of its genetic information, as DNA is subjected to 

unavoidable damage by Chemicals and radiation in the environment and 

by reactive molecules that are generated inside the cell. In this chapter, 

we describe the protein machines that replícate and repair the cell’s DNA. 

These machines catalyze some of the most rapid and accurate proc- 
esses that take place within cells, and the strategies they have evolved to 
achieve this feat are marvels of elegance and efficiency. 

Despite these Systems for protecting a cell's DNA frorn copying errors 
and accidental damage, permanent changes—or mutations —sometimes 
do occur. Although most mutations do not affect the organism in any 
noticeable way, some have profound consequences. Occasionally, these 
changes can benefit the organism: for example, mutations can make 
bacteria resistant to antibiotics that are used to kill them. What is more, 
changes in DNA sequence can produce small variations that underlie the 
differences between individuáis of the same species (Figure 6-1); when 
allowed to accumulate over millions of years, such changes provide the 
variety in genetic material that makes one species distinct frorn another, 
as we discuss in Chapter 9. 

But, mutations are much more likely to be detrimental than beneficial: in 
humans, they are responsible for thousands of genetic diseases, including 
cáncer. The survival of a cell or organism, therefore, depends on keeping 

LibertadDigital \ 2015 


CHAPTER 6 DNA Replication, Repair, and Recombination 

Figure 6-1 Genetic information is passed from one generation to 
the next. Differences in DNA can produce the variations that underlie 
the differences between individuáis of the same species—or, over 
time, the differences between one species and another. In this family 
photo, the children resemble one another and their parents more 
closely than they resemble other people because they inherit their 
genes from their parents. The cat shares many features with humans, 
but during the millions of years of evolution that have separated 
humans and cats, both have accumulated many changes in DNA that 
now make the two species different. The chicken is an even more 
distant relative. 

changes in its DNA to a mínimum. Without the protein machines that are 
continually monitoring and repairing damage to DNA, it is questionable 
whether life could exist at all. 


At each cell división, a cell must copy its genome with extraordinary 
accuracy. In this section, we explore how the cell achieves this feat, while 
duplicating its DNA at rates as high as 1000 nucleotides per second. 

Base-Pairing Enables DNA Replication 

In the preceding chapter, we saw that each strand of a DNA double helix 
contains a sequence of nucleotides that is exactly complementary to 
the nucleotide sequence of its partner strand. Each strand can therefore 
serve as a témplate, or mold, for the synthesis of a new complementary 
strand. In other words, if we desígnate the two DNA strands as S and S', 
strand S can serve as a témplate for making a new strand S', while strand 
S' can serve as a témplate for making a new strand S (Figure 6-2). Thus, 
the genetic information in DNA can be accurately copied by the beauti- 
fully simple process in which strand S separates from strand S', and each 
separated strand then serves as a témplate for the production of a new 
complementary partner strand that is identical to its former partner. 

The ability of each strand of a DNA molecule to act as a témplate for 
producing a complementary strand enables a cell to copy, or replícate, 
its genes before passing them on to its descendants. But the task is awe- 
inspiring, as it can involve copying billions of nucleotide pairs every time 
a cell divides. The copying must be carried out with incredible speed and 
accuracy: in about 8 hours, a dividing animal cell will copy the equivalent 
of 1000 books like this one and, on average, get no more than a few let- 
ters wrong. This impressive feat is performed by a cluster of proteins that 
together form a replication machine. 

Figure 6-2 DNA acts as a témplate 
for its own duplication. Because the 
nucleotide A will successfully pair only 
with T, and G with C, each strand of a 
DNA double helix—labeled here as 
the S strand and its complementary 
S' strand—can serve as a témplate to 
specify the sequence of nucleotides in its 
complementary strand. In this way, both 
strands of a DNA double helix can be 
copied precisely. 




r “ 



LibertadDigital \ 2015 

DNA Replication 199 

Figure 6-3 In each round of DNA replication, each of the two 
strands of DNA ¡s used as a témplate for the formation of a new, 
complementary strand. DNA replication is "semiconservative" 
because each daughter DNA double helix is composed of one 
conserved strand and one newly synthesized strand. 

DNA replication produces two complete double hélices from the original 
DNA molecule, with each new DNA helix being identical (except for rare 
copying errors) in nucleotide sequence to the original DNA double helix 
(see Figure 6-2). Because each parental strand serves as the témplate for 
one new strand, each of the daughter DNA double hélices ends up with 
one of the original (oíd) strands plus one strand that is completely new; 
this style of replication is said to be semiconservative (Figure 6-3). In How 
We Know, pp. 200-202, we discuss the experiments that first demon- 
strated that DNA is replicated in this way. 

DNA Synthesis Begins at Replication Origins 

The DNA double helix is normally very stable: the two DNA strands are 
locked together firmly by the large numbers of hydrogen bonds between 
the bases on both strands (see Figure 5-2). As a result, only temperatures 
approaching those of boiling water provide enough thermal energy to 
sepárate the two strands. To be used as a témplate, however, the double 
helix must first be opened up and the two strands separated to expose 
unpaired bases. How does this occur at the temperatures found in living 

The process of DNA synthesis is begun by initiator proteins that bind to 
specific DNA sequences called replication origins. Here, the initiator 
proteins pry the two DNA strands apart, breaking the hydrogen bonds 
between the bases (Figure 6-4). Although the hydrogen bonds collec- 
tively make the DNA helix veiy stable, individually each hydrogen bond 
is weak (as discussed in Chapter 2). Separating a short length of DNA a 
few base pairs at a time therefore does not require a large energy input, 
and the initiator proteins can readily unzip the double helix at normal 

In simple cells such as bacteria or yeast, replication origins span approxi- 
mately 100 nucleotide pairs. They are composed of DNA sequences that 
attract the initiator proteins and are especially easy to open. We saw in 
Chapter 5 that an A-T base pair is held together by fewer hydrogen bonds 
than is a G-C base pair. Therefore, DNA rich in A-T base pairs is relatively 
easy to pulí apart, and A-T-rich stretches of DNA are typically found at 
replication origins. 

A bacterial genome, which is typically contained in a circular DNA mol¬ 
ecule of several million nucleotide pairs, has a single replication origin. 
The human genome, which is very much larger, has approximately 10,000 
such origins—an average of 220 origins per chromosome. Beginning 
DNA replication at many places at once greatly shortens the time a cell 
needs to copy its entire genome. 

Once an initiator protein binds to DNA at a replication origin and locally 
opens up the double helix, it attracts a group of proteins that carry out 
DNA replication. These proteins form a replication machine, in which 
each protein carries out a specific function. 

Two Replication Forks Form at Each Replication Origin 

DNA molecules in the process of being replicated contain Y-shaped 
junctions called replication forks. Two replication forks are formed at 



i i 





A A A 



repllcatlon origin hellcal 
I l DNA v 

1 double helix opened 
with the aid of 
initiator proteins 

single-stranded DNAtemplates 
ready for DNA synthesis 

Figure 6-4 A DNA double helix is opened 
at replication origins. DNA sequences at 
replication origins are recognized by initiator 
proteins (not shown), which locally pry apart 
the two strands of the double helix. The 
exposed single strands can then serve as 
templates for copying the DNA. 

LibertadDigital \ 2015 




In 1953, James Watson and Francis Crick published 
their famous two-page paper describing a model for the 
structure of DNA (see Figure 5-2). In it, they proposed 
that complementary bases—adenine and thymine, gua- 
nine and cytosine—pair with one another along the 
center of the double helix, holding together the two 
strands of DNA. At the very end of this succinct scien- 
tific blockbuster, they comment, almost as an aside, "It 
has not escaped our notice that the specific pairing we 
have postulated immediately suggests a possible copy- 
ing mechanism for the genetic material." 

Indeed, one month after the classic paper appeared in 
print in the journal Nature, Watson and Crick published 
a second article, suggesting how DNA might be dupli- 
cated. In this paper, they proposed that the two strands 
of the double helix unwind, and that each strand serves 
as a témplate for the synthesis of a complementaiy 
daughter strand. In their model, dubbed semiconserva- 
tive replication, each new DNA molecule consists of one 
strand derived from the original parent molecule and 
one newly synthesized strand (Figure 6-5A). 

We now know that Watson and Crick's model for DNA 
replication was correct—but it was not universally 
accepted at first. Respected physicist-turned-geneticist 
Max Delbrück, for one, got hung up on what he termed 
"the untwiddling problem;" that is: how could the two 
strands of a double helix, twisted around each other 

so many times all along their great length, possibly be 
unwound without making a big tangled mess? Watson 
and Crick's conception of the DNA helix opening up like 
a zipper seemed, to Delbrück, physically unlikely and 
simply "too inelegant to be efflcient." 

Instead, Delbrück proposed that DNA replication pro- 
ceeds through a series of breaks and reunions, in which 
the DNA backbone is broken and the strands are cop- 
ied in short segments—perhaps only 10 nucleotides at 
a time—before being rejoined. In this model, which was 
later dubbed dispersive, the resulting copies would be 
patchwork collections of oíd and new DNA, each strand 
containing a mixture of both (Figure 6-5B). No unwind- 
ing was necessary. 

Yet a third camp promoted the idea that DNA replica¬ 
tion might be conservative: that the parent helix would 
somehow remain entirely intact after copying, and the 
daughter molecule would contain two entirely new DNA 
strands (Figure 6-5C). To determine which of these 
models was correct, an experiment was needed—one 
that would reveal the composition of the newly syn¬ 
thesized DNA strands. That's where Matt Meselson and 
Frank Stahl carne in. 

As a gradúate student working with Linus Pauling, 
Meselson was toying with a method for telling the differ- 
ence between oíd and new proteins. After chatting with 
Delbrück about Watson and Crick’s replication model, it 


Figure 6-5 Three models for DNA replication make different predictions. (A) In the semiconservative model, each parent strand 
serves as a témplate for the synthesis of a new daughter strand. The first round of replication would produce two hybrid molecules, 
each containing one strand from the original parent in addition to one newly synthesized strand. A subsequent round of replication 
would yield two hybrid molecules and two molecules that contain none of the original parent DNA (see Figure 6-3). (B) In the dispersive 
model, each generation of daughter DNA will contain a mixture of DNA from the parent strands and the newly synthesized DNA. 

(C) In the conservative model, the parent molecule remains intact after being copied. In this case, the first round of replication would 
yield the original parent double helix and an entirely new double helix. For each model, parent DNA molecules are shown in orange; 
newly replicated DNA is red. Note that only a very small segment of DNA is shown for each model. 

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DNA Replication 


occurred to Meselson that the approach he'd envisaged 
for exploring protein synthesis might also work for stud- 
ying DNA. In the summer of 1954, Meselson met Stahl, 
who was then a gradúate student in Rochester, NY, and 
they agreed to collaborate. It took a few years to get 
everything working, but the two eventually performed 
what has come to be known as "the most beautiful 
experiment in biology." 

Their approach, in retrospect, was stunningly straight- 
forward. They started by growing two batches of E. coli 
bacteria, one in a médium containing a heavy isotope 
of nitrogen, 15 N, the other in a médium containing the 
normal, lighter 14 N. The nitrogen in the nutrient médium 
gets incorporated into the nucleotide bases and, from 
there, makes its way into the DNA of the organism. 
After growing bacterial cultures for many generations in 
either the 1S N- or 14 N-containing médium, the research- 
ers had two flasks of bacteria, one whose DNA was 
heavy, the other whose DNA was light. Meselson and 
Stahl then broke open the bacterial cells and loaded the 
DNA into tubes containing a high concentration of the 
salt cesium chloride. When these tubes are centrifuged 
at high speed, the cesium chloride forms a density gra- 
dient, and the DNA molecules float or sink within the 
solution until they reach the point at which their density 
equals that of the surrounding salt solution (see Panel 
4-3, pp. 164-165). Using this method, called equilibrium 

density centrifugation, Meselson and Stahl found that 
they could distinguish between heavy ( 15 N-containing) 
DNA and light ( 14 N-containing) DNA by observing the 
positions of the DNA within the cesium chloride gradi- 
ent. Because the heavy DNA was denser than the light 
DNA, it collected at a position nearer to the bottom of 
the centrifuge tube (Figure 6-6). 

Once they had established this method for differentiat- 
ing between light and heavy DNA, Meselson and Stahl 
set out to test the various hypotheses proposed for DNA 
replication. To do this, they took a flask of bacteria that 
had been grown in heavy nitrogen and transferred the 
bacteria into a médium containing the light isotope. At 
the start of the experiment, all the DNA would be heavy. 
But, as the bacteria divided, the newly synthesized DNA 
would be light. They could then monitor the accumula- 
tion of light DNA and see which model, if any, best fit the 
data. After one generation of growth, the researchers 
found that the parental, heavy DNA molecules—those 
made of two strands containing 15 N—had disappeared 
and were replaced by a new species of DNA that banded 
at a density halfway between those of 15 N-DNA and 14 N- 
DNA (Figure 6-7). These newly synthesized daughter 
hélices, Meselson and Stahl reasoned, must be hybrids— 
containing both heavy and light isotopes. 

Right away, this observation ruled out the conserva- 
tive model of DNA replication, which predicted that 

^ t 

heavy 15 N-DNA forms a k light ,4 N-DNA forms a 

high-density band, closer /\\J low-density band, closer 

to the bottom of the tube r T to the top of the tube 

Figure 6-6 Centrifugation in a 
cesium chloride gradient allows 
the separation of heavy and light 
DNA. Bacteria are grown for several 
generations in a médium containing 
either 1S N (the heavy isotope) or 14 N 
(the light isotope) to label their DNA. 
The cells are then broken open, and the 
DNA ¡s loaded into an ultracentrifuge 
tube containing a cesium chloride salt 
solution. These tubes are centrifuged 
at high speed for two days to allow 
the DNAto collect in a región where 
its density matches that of the salt 
surrounding it. The heavy and light 
DNA molecules collect in different 
positions in the tube. 

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CHAPTER 6 DNA Replication, Repair, and Recombination 

light médium 

(B) bacteria grown in 
heavy médium 




(C) bacteria grown an 
additional 20 min in 
light médium 


DNA molecules of intermedíate weight 

Figure 6-7 The first part of the Meselson-Stahl experiment ruled out the 
conservative model of DNA replication. (A) Bacteria grown in light médium 
(containing 14 N) yield DNA that forms a band high up in the centrifuge tube, whereas 
bacteria grown in 15 N-containing heavy médium (B) produce DNA that migrates 
further down the tube. When bacteria grown in a heavy médium are transferred 
to a light médium and allowed to continué dividing, they produce a band whose 
position falls somewhere between that of the parent bands (C). These results rule 
out the conservative model of replication but do not distinguish between the 
semiconservative and dispersive models, both of which predict the formation of hybrid 
daughter DNA molecules. 

The fact that the results carne out looking so clean—with discrete bands forming 
at the expected positions for newly replicated hybrid DNA molecules—was a happy 
accident of the experimental protocol. The researchers used a hypodermicsyringe to 
load their DNA samples into the ultracentrifuge tubes (see Figure 6-6). In the process, 
they unwittingly sheared the large bacterial chromosome into smaller fragments. 

Had the chromosomes remained whole, the researchers might have ¡solated DNA 
molecules that were only partially replicated, because many cells would have been 
caught in the middle of copying their DNA. Molecules in such an intermedíate stage 
of replication would not have separated into such discrete bands. But because the 
researchers were instead working with smaller pieces of DNA, the likelihood that any 
given fragment had been fully replicated—and contained a complete parent and 
daughter strand—was high, thus yielding nice, clean results. 

the parental DNA would remain 
entirely heavy, while the daughter 
DNA would be entirely light (see 
Figure 6-5C). The data matched with 
the semiconservative model, which 
predicted the formation of hybrid 
molecules containing one strand of 
heavy DNA and one strand of light 
(see Figure 6-5A). The results, how- 
ever, were also consistent with the 
dispersive model, in which hybrid 
DNA strands would contain a mix¬ 
ture of heavy and light DNA (see 
Figure 6-5B). 

To distinguish between the two 
models, Meselson and Stahl tumed 
up the heat. When DNA is sub- 
jected to high temperature, the 
hydrogen bonds holding the two 
strands together break and the 
helix comes apart, leaving a collec- 
tion of single-stranded DNAs. When 
the researchers heated their hybrid 
molecules before centrifuging, they 
discovered that one strand of the 
DNA was heavy, whereas the other 
was light. This observation sup- 
ported only the semiconservative 
model; if the dispersive model were 
correct, the resulting strands, each 
containing a mottled assembly of 
heavy and light DNA, would have all 
banded together at an intermedíate 

According to historian Frederic 
Lawrence Holmes, the experiment 
was so elegant and the results 
so clean that Stahl—when being 
interviewed for a position at Yale 
University—was unable to fill the 50 
minutes allotted for his talk. "I was 
finished in 25 minutes," said Stahl, 
"because that is all it takes to tell that 
experiment. It's so totally simple and 
contained." Stahl did not get the job 
at Yale, but the experiment convinced 
biologists that Watson and Crick had 
been correct. In fact, the results were 
accepted so widely and rapidly that 
the experiment was described in a 
textbook before Meselson and Stahl 
had even published the data. 

LibertadDigital \ 2015 

DNA Replication 203 

each replication origin (Figure 6-8). At each fork, a replication machine 
moves along the DNA, opening up the two strands of the double helix 
and using each strand as a témplate to make a new daughter strand. The 
two forks move away from the origin in opposite directions, unzipping 
the DNA double helix and replicating the DNA as they go (Figure 6-9). 
DNA replication in bacterial and eukaiyotic chromosomes is therefore 
termed bidirecüonal. The forks move veiy rapidly—at about 1000 nucle- 
otide pairs per second in bacteria and 100 nucleotide pairs per second 
in humans. The slower rate of fork movement in humans (indeed, in all 
eukaiyotes) may be due to the difficulties in replicating DNA through the 
more complex chromatin structure of eukaryotic chromosomes. 

replication forks 
/ replication \ 

/ origin 1 

/ st \ ’CS 

- y, \\ - 

F V 

témplate DNA newly synthesized DNA 

Figure 6-8 DNA synthesis occurs at 
Y-shaped junctions called replication 
forks. Two replication forks are formed at 
each replication origin. 

DNA Polymerase Synthesizes DNA Using a Parental Strand 
as Témplate 

The movement of a replication fork is driven by the action of the replica¬ 
tion machine, at the heart of which is an enzyme called DNA polymerase 
This enzyme catalyzes the addition of nucleotides to the 3' end of a grow- 
ing DNA strand, using one of the original, parental DNA strands as a 
témplate. Base pairing between an incoming nucleotide and the témplate 
strand determines which of the four nucleotides (A, G, T, or C) will be 
selected. The final product is a new strand of DNA that is complementaiy 
in nucleotide sequence to the témplate (Figure 6-10). 

The polymerization reaction involves the formation of a phosphodiester 
bond between the 3' end of the growing DNA chain and the 5'-phosphate 
group of the incoming nucleotide, which enters the reaction as a dcoxy- 
ribonucleoside triphosphate. The energy for polymerization is provided 

Figure 6-9 The two replication forks move away ¡n opposite directions at 
each replication origin. (A) These drawings represent the same portion of a DNA 
molecule as it might appear at different times during replication. The orange 
lines represent the two parental DNA strands; the red lines represent the newly 
synthesized DNA strands. (B) An electrón micrograph showlng DNA replicating in 
an early fly embryo. The particles visible along the DNA are nucleosomes, structures 
made of DNA and the protein complexes around which the DNA is wrapped 
(discussed in Chapter 5). The chromosome in this micrograph is the one that was 
redrawn in sketch (2) above. (Electron micrograph courtesy of Victoria Foe.) 


Look carefully at the micrograph and 
drawing 2 in Figure 6-9. 

A. Using the scale bar, estímate the 
lengths of the DNA strands between 
the replication forks. Numbering the 
replication forks sequentially from 
the left, how long will it take until 
forks 4 and 5, and forks 7 and 8, 
respectively, collide with each other? 
(Recall that the distance between 
the bases in DNA is 0.34 nm, and 
eukaryotic replication forks move at 
about 100 nucleotides per second.) 
For this question, disregard the 
nucleosomes seen in the micrograph 
and assume that the DNA is fully 

B. The fly genome is about 

1.8 x 10 8 nucleotide pairs in size. 
What fraction of the genome is 
shown in the micrograph? 

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CHAPTER 6 DNA Replication, Repair, and Recombination 

témplate strand 

Figure 6-10 A new DNA strand is 
synthesized in the 5'-to-3' direction. 

At each step, the appropriate incoming 
nucleotide ¡s selected by forming base pairs 
with the next nucleotide ¡n the témplate 
strand: A with T, T with A, C with G, and G 
with C. Each is added to the 3' end of the 
growing new strand, as indicated. 

by the incoming deoxyribonucleoside triphosphate itself: hydrolysis 
of one of its high-energy phosphate bonds fuels the reaction that links 
the nucleotide monomer to the chain, releasing pyrophosphate (Figure 
6-11). Pyrophosphate is further hydrolyzed to inorganic phosphate (P¡), 
which makes the polymerization reaction effectively irreversible (see 
Figure 3-41). 

DNA polymerase does not dissociate from the DNA each time it adds a 
new nucleotide to the growing strand; rather, it stays associated with the 
DNA and moves along the témplate strand stepwise for many cycles of 
the polymerization reaction (Movie 6.1). We will see later that a special 
protein keeps the polymerase attached to the DNA, as it repeatedly adds 
new nucleotides to the growing strand. 

The Replication Fork Is Asymmetrical 

The 5'-to-3' direction of the DNA polymerization reaction poses a problem 
at the replication fork. As illustrated in Figure 5-2, the sugar-phosphate 
backbone of each strand of a DNA double helix has a unique Chemical 
direction, or polarity, determined by the way each sugar residue is linked 
to the next, and the two strands in the double helix are antiparallel; that 
is, they run in opposite directions. As a consequence, at each replica¬ 
tion fork, one new DNA strand is being made on a témplate that runs 
in one direction (3' to 5'), whereas the other new strand is being made 
on a témplate that runs in the opposite direction (5' to 3') (Figure 6-12). 
The replication fork is therefore asymmetrical. Looking at Figure 6-9A, 
however, it appears that both of the new DNA strands are growing in the 
same direction; that is, the direction in which the replication fork is mov- 
ing. That observation suggests that one strand is being synthesized in the 
5'-to-3' direction and the other in the 3'-to-5' direction. 

Figure 6-11 DNA polymerase adds a deoxyribonucleotide to the 3' end of a growing DNA chain. (A) Nucleotides enterthe 
reaction as deoxyribonucleoside triphosphates. This incoming nucleotide forms a base pair with its partner in the témplate strand. 

It is then linked to the free 3' hydroxyl on the growing DNA strand. The new DNA strand is therefore synthesized in the 5'-to-3' 
direction. Breakage of a high-energy phosphate bond in the incoming nucleoside triphosphate—accompanied by the release of 
pyrophosphate—provides the energy for the polymerization reaction. (B) The reaction is catalyzed by the enzyme DNA polymerase 
(light green ). The polymerase guides the incoming nucleotide to the témplate strand and positions it such that its 5' terminal 
phosphate will be able to react with the 3'-hydroxyl group on the newly synthesized strand. The gray arrow indicates the direction of 
polymerase movement. (C) Structure of DNA polymerase, as determined by X-ray crystallography, which shows the positioning of the 
DNA double helix. The témplate strand is the longer of the two DNA strands (Movie 6.1). 

LibertadDigital \ 2015 

DNA Replication 205 

Does the cell have two types of DNA polymerase, one for each direction? 
The answer is no: all DNA polymerases add new subunits only to the 3' 
end of a DNA strand (see Figure 6-11 A). As a result, a new DNA chain 
can be synthesized only in a 5'-to-3' direction. This can easily account 
for the synthesis of one of the two strands of DNA at the replication fork, 
but what happens on the other? This conundrum is solved by the use of 
a "backstitching" maneuver. The DNA strand that appears to grow in the 
incorrect 3'-to-5' direction is actually made discontinuously, in succes- 
sive, sepárate, small pieces—with the DNA polymerase moving backward 
with respect to the direction of replication-fork movement so that each 
new DNA fragment can be polymerized in the 5'-to-3' direction. 

The resulting small DNA pieces—called Okazaki fragments after the 
biochemists who discovered them—are later joined together to form a 
continuous new strand. The DNA strand that is made discontinuously in 
this way is called the lagging strand, because the backstitching imparts 
a slight delay to its synthesis; the other strand, which is synthesized con- 
tinuously, is called the leading strand (Figure 6-13). 

Although they differ in subtle details, the replication forks of all cells, 
prokaryotic and eukaryotic, have leading and lagging strands. This com- 
mon feature arises from the fact that all DNA polymerases work only in 
the 5'-to-3' direction—a restriction that provides cells with an important 
advantage, as we discuss next. 

newly synthesized 
5' ) 

direction of replication- 
fork movement 

Figure 6-12 At a replication fork, the two 
newly synthesized DNA strands are of 
opposite polarities. This is because the two 
témplate strands are oriented in opposite 

DNA Polymerase Is Self-correcting 

DNA polymerase is so accurate that it makes only about one error in 
every 10 7 nucleotide pairs it copies. This error rate is much lower than 
can be explained simply by the accuracy of complementary base-pairing. 
Although A-T and C-G are by far the most stable base pairs, other, less 
stable base pairs—for example, G-T and C-A—can also be formed. Such 
incorrect base pairs are formed much less frequently than correct ones, 
but, if allowed to remain, they would result in an accumulation of muta- 
tions. This disaster is avoided because DNA polymerase has two special 
qualities that greatly increase the accuracy of DNA replication. First, 
the enzyme carefully monitors the base-pairing between each incom- 
ing nucleotide and the témplate strand. Only when the match is correct 
does DNA polymerase catalyze the nucleotide-addition reaction. Second, 

Okazaki fragments 

direction of fork movement 

Figure 6-13 At each replication fork, the 
lagging DNA strand is synthesized in 
pieces. Because both of the new strands 
at a replication fork are synthesized in the 
5'-to-3' direction, the lagging strand of 
DNA must be made initially as a series of 
short DNA strands, which are later joined 
together. The upper diagram shows two 
replication forks moving in opposite 
directions; the lower diagram shows the 
same forks a short time later. To replícate 
the lagging strand, DNA polymerase uses 
a backstitching mechanism: it synthesizes 
short pieces of DNA (called Okazaki 
fragments) in the 5'-to-3' direction and then 
moves back along the témplate strand 
(toward the fork) before synthesizing the 
next fragment. 

LibertadDigital \ 2015 


CHAPTER 6 DNA Replication, Repair, and Recombination 

DNA polymerase 


5 ' 3' DNA strand 

5’ XX 


^ 3' 

I III’ . 







¡III 4 



Figure 6-14 During DNA synthesis, DNA 
polymerase proofreads its own work. If an 

¡ncorrect nucleotide ¡s added to a growing 
strand, the DNA polymerase cleaves ¡t from 
the strand and replaces it with the corred 
nucleotide before contlnulng. 

Figure 6-15 DNA polymerase contains 
sepárate sites for DNA synthesis and 
proofreading. The diagrams are based on 
the structure of an E. coli DNA polymerase 
molecule, as determined by X-ray 
crystallography. DNA polymerase is shown 
with the repllcating DNA molecule and the 
polymerase in the polymerizing mode ( left) 
and ¡n the proofreading mode (right). The 
catalytic sites for the polymerization activity 
(P) and error-correcting proofreading activity 
(E) are ¡ndlcated. When the polymerase 
adds an ¡ncorrect nucleotide, the newly 
syntheslzed DNA strand (red) transiently 
unpalrs from the témplate strand ( orange ), 
and ¡ts growing 3' end moves into the error- 
correcting catalytic site (E)to be removed. 

when DNA polymerase makes a rare mistake and adds the wrong nucle¬ 
otide, it can correct the error through an activity called proofreading. 
Proofreading takes place at the same time as DNA synthesis. Before the 
enzyme adds the next nucleotide to a growing DNA strand, it checks 
whether the previously added nucleotide is correctly base-paired to the 
témplate strand. If so, the polymerase adds the next nucleotide; if not, 
the polymerase clips off the mispaired nucleotide and tries again (Figure 
6-14). This proofreading is carried out by a nuclease that cleaves the 
phosphodiester backbone. Polymerization and proofreading are tightly 
coordinated, and the two reactions are carried out by different catalytic 
domains in the same polymerase molecule (Figure 6-15). 

This proofreading mechanism explains why DNA polymerases synthesize 
DNA only in the 5'-to-3' direction, despite the need that this imposes for a 
cumbersome backstitching mechanism at the replication fork (see Figure 
6-13). A hypothetical DNA polymerase that synthesized in the 3'-to-5' 
direction (and would thereby circumvent the need for backstitching) 
would be unable to proofread: if it removed an incorrectly paired nucle¬ 
otide, the polymerase would create a Chemical dead end—a chain that 
could no longer be elongated. Thus, for a DNA polymerase to function 
as a self-correcting enzyme that removes its own polymerization errors 
as it moves along the DNA, it must proceed only in the 5'-to-3' direction. 

Short Lengths of RNA Act as Primers for DNA Synthesis 

We have seen that the accuracy of DNA replication depends on the 
requirement of the DNA polymerase for a correctly base-paired 3' end 
before it can add more nucleotides to a growing DNA strand. How then 
can the polymerase begin a completely new DNA strand? To get the 
process started, a different enzyme is needed—one that can begin a new 
polynucleotide strand simply by joining two nucleotides together without 
the need for a base-paired end. This enzyme does not, however, syn¬ 
thesize DNA. It makes a short length of a closely related type of nucleic 
acid—RNA (ribonucleic acid)—using the DNA strand as a témplate. This 
short length of RNA, about 10 nucleotides long, is base-paired to the tém¬ 
plate strand and provides a base-paired 3' end as a starting point for DNA 
polymerase. It thus serves as a primer for DNA synthesis, and the enzyme 
that synthesizes the RNA primer is known as primase. 

Primase is an example of an RNA polymerase, an enzyme that synthesizes 
RNA using DNA as a témplate. A strand of RNA is very similar chemically 
to a single strand of DNA except that it is made of ribonucleotide subu- 
nits, in which the sugar is ribose, not deoxyribose; RNA also differs from 
DNA in that it contains the base uracil (U) instead of thymine (T) (see 
Panel 2-6, pp. 76-77). However, because U can form a base pair with 
A, the RNA primer is synthesized on the DNA strand by complementary 
base-pairing in exactly the same way as is DNA (Figure 6-16). 





LibertadDigital \ 2015 

DNA Replication 207 

Figure 6-16 RNA primers are synthesized by an RNA polymerase 
called primase, which uses a DNA strand as a témplate. Like DNA 
polymerase, primase works ¡n the 5'-to-3' direction. Unlike DNA 
polymerase, however, primase can start a new polynucleotide chain by 
joining together two nucleoside triphosphates without the need for 
a base-paired 3' end as a starting point. (In this case, ribonucleoside 
triphosphates, ratherthan deoxyribonucleoside triphosphates, provide 
the incoming nucleotides.) 

* < 

5 , ¿ mmmmmmmmmmmmm U3 , 

For the leading strand, an RNA primer is needed only to start replica¬ 
tion at a replication origin; once a replication fork has been established, 
the DNA polymerase is continuously presented with a base-paired 3' 
end as it tracks along the témplate strand. But on the lagging strand, 
where DNA synthesis is discontinuous, new primers are needed to 
keep polymerization going (see Figure 6-13). The movement of the 
replication fork continually exposes unpaired bases on the lagging 
strand témplate, and new RNA primers are laid down at intervals along 
the newly exposed, single-stranded stretch. DNA polymerase adds a 
deoxyribonucleotide to the 3' end of each primer to start a new Okazaki 
fragment, and it will continué to elongate this fragment until it runs into 
the next RNA primer (Figure 6-17). 

To produce a continuous new DNA strand frorn the many sepárate pieces 
of nucleic acid made on the lagging strand, three additional enzymes are 
needed. These act quickly to remove the RNA primer, replace it with DNA, 
and join the DNA fragments together. Thus, a nuclease degrades the RNA 
primer, a DNA polymerase called a repair polymerase then replaces this 
RNA with DNA (using the end of the adjacent Okazaki fragment as a 
primer), and the enzyme DNA ligase joins the 5'-phosphate end of one 
DNA fragment to the adjacent 3'-hydroxyl end of the next (Figure 6-18). 
Primase can begin new polynucleotide chains, but this activity is possible 
because the enzyme does not proofread its work. As a result, primers fre- 
quently contain mistakes. But because primers are made of RNA instead 
of DNA, they stand out as "suspect copy" to be automatically removed 
and replaced by DNA. The repair DNA polymerases that make this DNA, 
like the replicative polymerases, proofread as they synthesize. In this 
way, the cell’s replication machineiy is able to begin new DNA chains 
and, at the same time, ensure that all of the DNA is copied faithfully. 

Proteins at a Replication Fork Cooperate to Form 
a Replication Machine 

DNA replication requires the cooperation of a large number of proteins 
that act in concert to open up the double helix and synthesize new DNA. 
These proteins form part of a remarkably complex replication machine. 
The first problem faced by the replication machine is accessing the 

3' HO^. 

. 1 


RNA primer primase 






oíd RNA 

new RNA primer 
synthesis by 

DNA I DNA polymerase adds nucleotides 

lagging- to 3 ' enc) of new RNA primer 
strand to start new Okazaki fragment 
témplate t 

DNA polymerase finishes 
DNA fragment 

Figure 6-17 Múltiple enzymes are required to synthesize Okazaki 
fragments on the lagging DNA strand. In eukaryotes, RNA primers 
are made at ¡ntervals of about 200 nucleotides on the lagging strand, 
and each RNA primer is approximately 10 nucleotides long. Primers 
are removed by nucleases that recognize an RNA strand in an RNA/ 
DNA helix and degrade it; this leaves gaps that are filled in by a 
repair DNA polymerase that can proofread as it filis in the gaps. The 
completed fragments are finally joined together by an enzyme called 
DNA ligase, which catalyzes the formation of a phosphodiester bond 
between the 3'-OH end of one fragment and the 5'-phosphate end of 
the next, thus linking up the sugar-phosphate backbones. This nick- 
sealing reaction requires an input of energy in the form of ATP (not 
shown; see Figure 6-18). 

nick sealed by DNA ligase, 
joining new Okazaki fragment 
to the growing DNA strand 

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CHAPTER 6 DNA Replication, Repair, and Recombination 

Figure 6-18 DNA ligase joins together 
Okazaki fragments on the lagging strand 
during DNA synthesis. The ligase enzyme 
uses a molecule of ATP to actívate the 5' 
end of one fragment (step 1) before forming 
a new bond with the 3' end of the other 
fragment (step 2). 

nucleotides that lie at the center of the helix. For DNA replication to occur, 
the double helix must be unzipped ahead of the replication fork so that 
the incoming nucleoside triphosphates can form base pairs with each 
témplate strand. Two types of replication proteins —DNA helicases and 
single-strand DNA-binding proteins —cooperate to carry out this task. The 
helicase sits at the very front of the replication machine where it uses the 
energy of ATP hydrolysis to propel itself forward, prying apart the double 
helix as it speeds along the DNA (Figure 6-19A and Movie 6.2). Single- 
strand DNA-binding proteins cling to the single-stranded DNA exposed 
by the helicase, transiently preventing the strands from re-forming base 
pairs and keeping them in an elongated form so that they can serve as 
efficient templates. 

Figure 6-19 DNA synthesis ¡s carried 
out by a group of proteins that act 
together as a replication machine. 

(A) DNA polymerases are held on 
the leading and lagging strands by 
circular protein clamps that allow the 
polymerases to slide. On the lagging- 
strand témplate, the clamp detaches 
each time the polymerase completes an 
Okazaki fragment. A clamp loader (not 
shown) ¡s required to attach a sliding 
clamp each time a new Okazaki fragment 
¡s begun. At the head of the fork, a DNA 
helicase unwinds the strands ofthe 
parental DNA double helix. Single-strand 
DNA-binding proteins keep the DNA 
strands apart to provide access for the 
primase and polymerase. For simplicity, 
this diagram shows the proteins working 
¡ndependently; in the cell, they are held 
together in a large replication machine, 
as shown in (B). 

(B) This diagram shows a current view of 
how the replication proteins are arranged 
when a replication fork ¡s moving. To 
generate this structure, the lagging 
strand shown in (A) has been folded to 
bring its DNA polymerase in contad with 
the leading-strand DNA polymerase. This 
folding process also brings the 3' end 
of each completed Okazaki fragment 
cióse to the start site for the next Okazaki 
fragment. Because the lagging-strand 
DNA polymerase ¡s bound to the rest 
ofthe replication proteins, it can be 
reused to synthesize successive Okazaki 
fragments; in this diagram, the lagging- 
strand DNA polymerase is about to let go 
of its completed Okazaki fragment and 
move to the RNA primer that is being 
synthesized by the nearby primase. To 
watch the replication complex in action, 
see Movies 6.4 and 6.5. 

This localized unwinding of the DNA double helix itself presents a prob- 
lem. As the helicase pries open the DNA within the replication fork, the 

LibertadDigital \ 2015 

DNA Replication 209 

DNA on the other side of the fork gets wound more tightly. This excess 
twisting in front of the replication fork creates tensión in the DNA that—if 
allowed to build—makes unwinding the double helix increasingly diffi- 
cult and impedes the forward movement of the replication machinery 
(Figure 6-20A). Cells use proteins called DNA topoisomerases to relieve 
this tensión. These enzymes produce transient nicks in the DNA back- 
bone, which temporarily release the tensión; they then reseal the nick 
before falling off the DNA (Figure 6-20B). 

An additional replication protein, called a sliding clamp, keeps DNA 
polymerase firmly attached to the témplate while it is synthesizing new 
strands of DNA. Left on their own, most DNA polymerase molecules will 
synthesize only a short string of nucleotides before falling off the DNA 
témplate strand. The sliding clamp forms a ring around the newly formed 
DNA double helix and, by tightly gripping the polymerase, allows the 
enzyme to move along the témplate strand without falling off as it syn- 
thesizes new DNA (see Figure 6-19A and Movie 6.3). 

Assembly of the clamp around DNA requires the activity of another repli¬ 
cation protein, the clamp loader, which hydrolyzes ATP each time it locks 
a sliding clamp around a newly formed DNA double helix. This loading 
needs to occur only once per replication cycle on the leading strand; on 
the lagging strand, however, the clamp is removed and then reattached 
each time a new Okazaki fragment is made. 

Most of the proteins involved in DNA replication are held together in 
a large multienzyme complex that moves as a unit along the parental 
DNA double helix, enabling DNA to be synthesized on both strands in a 
coordinated manner. This complex can be likened to a miniature sewing 
machine composed of protein parts and powered by nucleoside triphos- 
phate hydrolysis (Figure 6-19B and Movies 6.4 and 6.5). 

Telomerase Replicates the Ends of Eukaryotic 

Having discussed how DNA replication begins at origins and how move¬ 
ment of a replication fork proceeds, we now turn to the special problem 

Figure 6-20 DNA topoisomerases 
relieve the tensión that builds up in 
front of a replication fork. (A) As DNA 

helicase unwinds the DNA double helix, 

¡t generates a section of overwound 
DNA. Tensión builds up because the 
chromosome is too large to rotate fast 
enough to relieve the buildup of torsional 
stress. The broken bars in the left-hand 
panel represent approxlmately 20 turns 
of DNA. (B) DNA topoisomerases relieve 
this stress by generating temporary nicks 
in the DNA. 


Discuss the following statement: 
"Primase is a sloppy enzyme that 
makes many mistakes. Eventually, 
the RNA primers it makes are 
disposed of and replaced with DNA 
synthesized by a polymerase with 
higher fidelity. This is wasteful. It 
would be more energy-efficient if a 
DNA polymerase made an accurate 
copy in the first place." 

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DNA Replication, Repair, and Recombination 

lagging strand 

leading strand 




leadlng strand 


lagging strand 

leading strand 

Figure 6-21 Without a special mechanism to replicate the ends of linear 
chromosomes, DNA would be lost during each round of cell división. DNA 

synthesis begins at origins of replication and continúes until the replication 
machinery reaches the ends of the chromosome. The leading strand ¡s reproduced ¡n 
its entirety. But the ends of the lagging strand can't be completed, because once the 
final RNA primer has been removed there ¡s no way to replace ¡t with DNA. These 
gaps at the ends of the lagging strand must be filled in by a special mechanism to 
keep the chromosome ends from shrinking with each cell división. 


A gene encoding one of the proteins 
involved in DNA replication has 
been inactivated by a mutation in a 
cell. In the absence of this protein, 
the cell attempts to replicate its 
DNA. What would happen during 
the DNA replication process ¡f 
each of the following proteins were 

A. DNA polymerase 

B. DNA ligase 

C. Sliding clamp for DNA 

D. Nuclease that removes RNA 

E. DNA helicase 

F. Primase 

of replicating the veiy ends of chromosomes. As we discussed previ- 
ously, because DNA replication proceeds only in the 5'-to-3' direction, 
the lagging strand of the replication fork has to be synthesized in the 
form of discontinuous DNA fragments, each of which is primed with an 
RNA primer laid down by a primase (see Figure 6-17). A serious problem 
arises, however, as the replication fork approaches the end of a chromo¬ 
some: although the leading strand can be replicated all the way to the 
chromosome tip, the lagging strand cannot. When the final RNA primer 
on the lagging strand is removed, there is no way to replace it (Figure 
6-21). Without a strategy to deal with this problem, the lagging strand 
would become shorter with each round of DNA replication; after repeated 
cell divisions, chromosomes would shrink—and eventually lose valuable 
genetic information. 

Bacteria solve this "end-replication" problem by having circular DNA 
molecules as chromosomes. Eukaryotes solve it by having long, repeti- 
tive nucleotide sequences at the ends of their chromosomes which are 
incorporated into structures called telomeres. These telomeric DNA 
sequences attract an enzyme called telomerase to the chromosome 
ends. Using an RNA témplate that is part of the enzyme itself, telomerase 
extends the ends of the replicating lagging strand by adding múltiple cop¬ 
ies of the same short DNA sequence to the témplate strand. This extended 
témplate allows replication of the lagging strand to be completed by con- 
ventional DNA replication (Figure 6-22). 

In addition to allowing replication of chromosome ends, telomeres form 
structures that mark the true ends of a chromosome. This allows the cell 
to distinguish unambiguously between the natural ends of chromosomes 
and the double-strand DNA breaks that sometimes occur accidentally in 

LibertadDigital \ 2015 

DNA Repair 







chromosome telomere repeat sequence 

5 ' x incomplete, newly synthesized lagging strand 



telomerase with its bound RNA témplate 

extended témplate strand 

-témplate of lagging strand 

^ telomere 
DNA synthesls 

directlon of 

Figure 6-22 Telomeres and telomerase 
prevent linear eukaryotic chromosomes 
from shortening with each cell división. 

For clarity, only the témplate DNA ( orange ) 
and newly synthesized DNA (red) of the 
lagging strand are shown (see bottom of 
Figure 6-21). To complete the replication 
of the lagging strand at the ends of a 
chromosome, the témplate strand is first 
extended beyond the DNA that is to 
be copied. To achieve this, the enzyme 
telomerase adds more repeats to the 
telomere repeat sequences at the 3' end of 
the témplate strand, which then allows the 
lagging strand to be completed by DNA 
polymerase, as shown. The telomerase 
enzyme carries a short piece of RNA (b/ue) 
with a sequence that is complementary to 
the DNA repeat sequence; this RNA acts as 
the témplate for telomere DNA synthesis. 
After the lagging-strand replication 
is complete, a short stretch of single- 
stranded DNA remains at the ends of the 
chromosome, as shown. To see telomerase 
in action, view Movie 6.6. 

the middle of chromosomes. These breaks are dangerous and must be 
immediately repaired, as we see in the next section. 


The diversity of living organisms and their success in colonizing almost 
every part of the Earth's surface depend on genetic changes accumulated 
gradually over millions of years. Some of these changes allow organisms 
to adapt to changing conditions and to thrive in new habitats. However, 
in the short term, and from the perspective of an individual organism, 
genetic alterations can be detrimental. In a multicellular organism, such 
permanent changes in the DNA—called mutations—can upset the organ- 
ism's extremely complex and finely tuned development and physiology. 
To survive and reproduce, individuáis must be genetically stable. This 
stability is achieved not only through the extremely accurate mechanism 
for replicating DNA that we have just discussed, but also through the 
work of a variety of protein machines that continually sean the genome 
for damage and fix it when it occurs. Although some changes arise from 
rare mistakes in the replication process, the majority of DNA damage is 
an unintended consequence of the vast number of Chemical reactions 
that occur inside cells. 

Most DNA damage is only temporary, because it is immediately corrected 
by processes collectively called DNA repair. The importance of these DNA 
repair processes is evident from the consequences of their malfunction. 
Humans with the genetic disease xeroderma pigmentosum, for example, 
cannot mend the damage done by ultraviolet (UV) radiation because they 
have inherited a detective gene for one of the proteins involved in this 
repair process. Such individuáis develop severe skin lesions, including 
skin cáncer, because of the accumulation of DNA damage in cells that 
are exposed to sunlight and the consequent mutations that arise in these 

In this section, we describe a few of the specialized mechanisms cells 
use to repair DNA damage. We then consider examples of what happens 
when these mechanisms fail—and discuss how the fidelity of DNA repli¬ 
cation and repair are reflected in our genome. 

LibertadDigital \ 2015 


CHAPTER 6 DNA Replication, Repair, and Recombination 

Figure 6-23 Depurination and 
deamination are the most frequent 
Chemical reactions known to create 
serious DNA damage in cells. 

(A) Depurination can remove guanine 
(or adenine) from DNA. (B) The major 
type of deamination reaction converts 
cytosine to an altered DNA base, uracil; 
however, deamination can also occur on 
other bases as well. Both depurination and 
deamination take place on double-helical 
DNA, and neither breakthe phosphodiester 






DNA strand 



Discuss the following statement: 
"The DNA repair enzymes that 
fix deamination and depurination 
damage must preferentially 
recognize such damage on newly 
synthesized DNA strands." 

DNA Damage Occurs Continually ¡n Cells 

Just like any other molecule in the cell, DNA is continually undergoing 
thermal collisions with other molecules, often resulting in major Chemi¬ 
cal changes in the DNA. For example, during the time it takes to read 
this sentence, a total of about a trillion (10 12 ) purine bases (A and G) 
will be lost from DNA in the cells of your body by a spontaneous reac¬ 
tion called depurination (Figure 6-23A). Depurination does not break the 
DNA phosphodiester backbone but instead removes a purine base from a 
nucleotide, giving rise to lesions that resemble missing teeth (see Figure 
6-25B). Another common reaction is the spontaneous loss of an amino 
group (deamination) from a cytosine in DNA to produce the base uracil 
(Figure 6-23B). Some chemically reactive by-products of cell metabolism 
also occasionally react with the bases in DNA, altering them in such a 
way that their base-pairing properties are changed. 

The ultraviolet radiation in sunlight is also damaging to DNA; it promotes 
covalent linkage between two adjacent pyrimidine bases, forming, for 
example, the thymine dimer shown in Figure 6-24. It is the failure to 
repair thymine dimers that spells trouble for individuáis with the disease 
xeroderma pigmentosum. 

Figure 6-24 The ultraviolet radiation 
in sunlight can cause the formation of 
thymine dimers. Two adjacent thymine 
bases have become covalently attached 
to each other to form a thymine dimer. 
Skin cells that are exposed to sunlight 
are especially susceptible to this type of 
DNA damage. 

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DNA Repair 



oíd strand 

| new strand 

deaminated C 

n A 



oíd strand 


oíd strand 

| new strand 

oíd strand 



These are only a few of many Chemical changes that can occur in our 
DNA. If left unrepaired, many of them would lead either to the substitu- 
tion of one nucleotide pair for another as a result of incorrect base-pairing 
during replication (Figure 6-25A) or to deletion of one or more nucleotide 
pairs in the daughter DNA strand after DNA replication (Figure 6-25B). 
Some types of DNA damage (thymine dimers, for example) can stall the 
DNA replication machinery at the site of the damage. 

In addition to this Chemical damage, DNA can also be altered by repli¬ 
cation itself. The replication machinery that copies the DNA can—quite 
rarely—incorpórate an incorrect nucleotide that it fails to correct via 
proofreading (see Figure 6-14). 

For each of these forms of DNA, cells possess a mechanism for repair, as 
we discuss next. 

Cells Possess a Variety of Mechanisms for Repairing DNA 

The thousands of random Chemical changes that occur every day in the 
DNA of a human cell—through thermal collisions or exposure to reac¬ 
tive metabolic by-products, DNA-damaging Chemicals, or radiation—are 
repaired by a variety of mechanisms, each catalyzed by a different set 
of enzymes. Nearly all these repair mechanisms depend on the dou- 
ble-helical structure of DNA, which provides two copies of the genetic 
information—one in each strand of the double helix. Thus, if the sequence 
in one strand is accidentally damaged, information is not lost irretriev- 
ably, because a backup versión of the altered strand remains in the 
complementary sequence of nucleotides in the other strand. Most DNA 
damage creates structures that are never encountered in an undamaged 
DNA strand; thus the good strand is easily distinguished from the bad. 
The basic pathway for repairing damage to DNA, illustrated schematically 
in Figure 6-26, involves three basic steps: 

1. The damaged DNA is recognized and removed by one of a variety 
of mechanisms. These involve nucleases, which cleave the cova- 
lent bonds that join the damaged nucleotides to the rest of the DNA 
strand, leaving a small gap on one strand of the DNA double helix 
in the región. 

2. A repair DNA polymerase binds to the 3'-hydroxyl end of the cut 
DNA strand. It then filis in the gap by making a complementary 
copy of the information stored in the undamaged strand. Although 

Figure 6-25 Chemical modifications of 
nucleotides, ¡f left unrepaired, produce 
mutations. (A) Deamination of cytosine, 
if uncorrected, results in the substitution 
of one base for another when the DNA 
is replicated. As shown in Figure 6-23B, 
deamination of cytosine produces uracil. 
Uracil differs from cytosine in its base- 
pairing properties and preferentially 
base-pairs with adenine. The DNA 
replication machinery therefore inserts 
an adenine when it encounters a uracil 
on the témplate strand. (B) Depurination, 
if uncorrected, can lead to the loss of 
a nucleotide pair. When the replication 
machinery encounters a missing purine 
on the témplate strand, it can skip to the 
next complete nucleotide, as shown, thus 
producing a daughter DNA molecule that 
is missing one nucleotide pair. In other 
cases (not shown), the replication machinery 
places an incorrect nucleotide across from 
the missing base, again resulting in a 

LibertadDigital \ 2015 


CHAPTER 6 DNA Replication, Repair, and Recombination 

Figure 6-26 The basic mechanism of 
DNA repair involves three steps. In step 1 
(excisión), the damage is cut out by one of 
a series of nucleases, each specialized for a 
type of DNA damage. In step 2 (resynthesis), 
the original DNAsequence ¡s restored by 
a repair DNA polymerase, which filis ¡n the 
gap created by the excisión events. In step 
3 (ligation), DNA ligase seáis the nick left 
in the sugar-phosphate backbone of the 
repalred strand. Nickseallng, which requires 
energy from ATP hydrolysis, remakes the 
broken phosphodlester bond between the 
adjacent nucleoides (see Figure 6-18). 

different from the DNA polymerase that replicates DNA, repair 
DNA polymerases synthesize DNA strands in the same way. For 
example, they elongate chains in the 5'-to-3' direction and have 
the same type of proofreading activity to ensure that the témplate 
strand is copied accurately. In many cells, this is the same enzyme 
that filis in the gap left after the RNA primers are removed during 
the normal DNA replication process (see Figure 6-17). 

3. When the repair DNA polymerase has filled in the gap, a break 
remains in the sugar-phosphate backbone of the repaired strand. 
This nick in the helix is sealed by DNA ligase, the same enzyme 
that joins the Okazaki fragments during replication of the lagging 
DNA strand. 

Steps 2 and 3 are nearly the same for most types of DNA damage, 
including the rare errors that arise during DNA replication. However, 
step 1 uses a series of different enzymes, each specialized for removing 
different types of DNA damage. Humans produce hundreds of different 
proteins that function in DNA repair. 

A DNA Mismatch Repair System Removes Replication 
Errors That Escape Proofreading 

Although the high fidelity and proofreading abilities of the cell's replica¬ 
tion machineiy generally prevent replication errors from occurring, rare 
mistakes do happen. Fortunately, the cell has a backup System—called 
mismatch repair—which is dedicated to correcting these errors. The 
replication machine makes approximately one mistake per 10 7 nucle- 
otides copied; DNA mismatch repair corrects 99% of these replication 
errors, increasing the overall accuracy to one mistake in 10 9 nucleotides 
copied. This level of accuracy is much, much higher than that generally 
encountered in our day-to-day lives (Table 6-1). 

Whenever the replication machinery makes a copying mistake, it leaves 
behind a mispaired nucleotide (commonly called a mismatch). If left 
uncorrected, the mismatch will result in a permanent mutation in the 
next round of DNA replication (Figure 6-27). A complex of mismatch 
repair proteins recognizes such a DNA mismatch, removes a portion of 
the DNA strand containing the error, and then resynthesizes the missing 
DNA. This repair mechanism restores the correct sequence (Figure 6-28). 
To be effective, the mismatch repair system must be able to recognize 
which of the DNA strands contains the error. Removing a segment 
from the strand of DNA that contains the correct sequence would only 


US Postal Service on-time delivery of local 
flrst-class malí 

13 late deliveries per 100 pareéis 

Alrllne luggage system 

1 lost bag per 150 

A professional typist typing at 120 words 
per minute 

1 mistake per 250 characters 

Driving a car ¡n the United States 

1 death per 10 4 people per year 

DNA replication (wlthout proofreading) 

1 mistake per 10 5 nucleotides 

DNA replication (wlth proofreading; 
without mismatch repair) 

1 mistake per 10 7 nucleotides 

DNA replication (wlth mismatch repair) 

1 mistake per 10 9 nucleotides 

LibertadDigital \ 2015 

DNA Repair 



parent DNA 


original parent strand 

original parent strand 

compound the mistake. The way the mismatch system solves this prob- 
lem is by always removing a portion of the newly made DNA strand. In 
bacteria, newly synthesized DNA lacks a type of Chemical modification 
that is present on the preexisting parent DNA. Other cells use other strat- 
egies for distinguishing their parent DNA from a newly replicated strand. 
Mismatch repair plays an important role in preventing cáncer. An inher- 
ited predisposition to certain cancers (especially some types of colon 
cáncer) is caused by mutations in genes that encode mismatch repair 
proteins. Humans inherit two copies of these genes (one from each par¬ 
ent), and individuáis who inherit one damaged mismatch repair gene 
are unaffected until the undamaged copy of the same gene is randomly 
mutated in a somatic cell. This mutant cell—and all of its progeny—are 
then deficient in mismatch repair; they therefore accumulate mutations 
more rapidly than do normal cells. Because cancers arise from cells that 
have accumulated múltiple mutations, a cell deficient in mismatch repair 
has a greatly enhanced chance of becoming cancerous. Thus, inheriting 
a damaged mismatch repair gene strongly predisposes an individual to 



original parent strand 

Figure 6-27 Errors made during DNA 
replication must be corrected to avoid 
mutations. If uncorrected, a mismatch will 
lead to a permanent mutation in one of the 
two DNA molecules produced by the next 
round of DNA replication. 

Double-Strand DNA Breaks Require a Different Strategy 
for Repair 

The repair mechanisms we have discussed thus far rely on the genetic 
redundancy built into every DNA double helix. If nucleotides on one 
strand are damaged, they can be repaired using the information present 
in the complementary strand. 

But what happens when both strands of the double helix are damaged 
at the same time? Radiation, mishaps at the replication fork, and vari- 
ous Chemical assaults can all fracture the backbone of DNA, creating a 




original parent strand 

Figure 6-28 Mismatch repair eliminates 
replication errors and restores the 
original DNA sequence. When mistakes 
occur during DNA replication, the repair 
machinery must replace the ¡ncorrect 
nucleotide on the newly synthesized strand, 
using the original parent strand as its 
témplate. This mechanism eliminates the 

original parent strand 

LibertadDigital \ 2015 


CHAPTER 6 DNA Replication, Repair, and Recombination 

Figure 6-29 Cells can repair 
double-strand breaks in one of 
two ways. (A) In nonhomologous 
end joining, the break ¡s first 
"cleaned" by a nuclease that 
chews back the broken ends 
to produce flush ends. The 
flush ends are then stitched 
together by a DNA ligase. Some 
nucleotides are lost in the repair 
process, as ¡ndicated by the black 
lines ¡n the repalred DNA. (B) If 
a double-strand break occurs in 
one of two daughter DNA double 
hélices after DNA replication 
has occurred, but before the 
daughter chromosomes have 
been separated, the undamaged 
double hellx can be readlly 
used as a témplate to repair 
the damaged double hellx by 
homologous recombination. Thls 
is a more involved process than 
non-homologous end joining, but 
it accurately restores the original 
DNA sequence at the site of the 
break. The detailed mechanism is 
presented in Figure 6-30. 


^accidental double-strand break 

5' 3> 5'31damaged 

3'5' J DNA molecule 


i3'-i undamaged 
■5'J DNA molecule 








deletion of DNA sequence 



double-strand break. Such lesions are particularly dangerous, because 
they can lead to the fragmentation of chromosomes and the subsequent 
loss of genes. 

This type of damage is especially difficult to repair. Each chromosome 
contains unique information; if a chromosome undergoes a double- 
strand break, and the broken pieces become separated, the cell has no 
spare copy it can use to reconstruct the information that is now missing. 
To handle this potentially disastrous type of DNA damage, cells have 
evolved two basic strategies. The first involves rapidly sticking the broken 
ends back together, before the DNA fragments drift apart and get lost. 
This repair mechanism, called nonhomologous end joining, occurs in 
many cell types and is carried out by a specialized group of enzymes that 
"clean" the broken ends and rejoin them by DNA ligation. This "quick and 
dirty" mechanism rapidly repairs the damage, but it comes with a price: 
in "cleaning" the break to make it ready for ligation, nucleotides are often 
lost at the site of repair (Figure 6-29A). 

In most cases, this emergency repair mechanism mends the damage 
without creating any additional problems. But if the imperfect repair dis- 
rupts the activity of a gene, the cell could suffer serious consequences. 
Thus, nonhomologous end joining can be a risky strategy for fixing 
broken chromosomes. So cells have an altemative, error-free strategy 
for repairing double-strand breaks, called homologous recombination 
(Figure 6-29B), as we discuss next. 

Homologous Recombination Can Flawlessly Repair DNA 
Double-Strand Breaks 

The problem with repairing a double-strand break, as we mentioned, 
is finding an intact témplate to guide the repair. However, if a double- 
strand break occurs in one double helix shortly after a stretch of DNA 
has been replicated, the undamaged double helix can readily serve as 
a témplate to guide the repair of the broken DNA: information on the 
undamaged strand of the intact double helix is used to repair the com- 
plementary broken strand in the other. Because the two DNA molecules 

LibertadDigital \ 2015 

DNA Repair 


are homologous—they have identical nucleotide sequences outside the 
broken región—this mechanism is known as homologous recombina- 
tion. It results in a flawless repair of the double-strand break, with no loss 
of genetic information (see Figure 6-29B). 

Homologous recombination most often occurs shortly after a cell's DNA 
has been replicated before cell división, when the duplicated hélices are 
still physically cióse to each other (Figure 6-30A). To initiate the repair, a 
nuclease chews back the 5' ends of the two broken strands at the break 
(Figure 6-30B). Then, with the help of specialized enzymes, one of the 
broken 3' ends "invades" the unbroken homologous DNA dúplex and 
searches for a complementary sequence through base-pairing (Figure 
6-30C). Once an extensive, accurate match is found, the invading strand 
is elongated by a repair DNA polymerase, using the complementary strand 
as a témplate (Figure 6-30D). After the repair polymerase has passed 
the point where the break occurred, the newly repaired strand rejoins 
its original partner, forming base pairs that hold the two strands of the 
broken double helix together (Figure 6-30E). Repair is then completed 
by additional DNA synthesis at the 3' ends of both strands of the broken 
double helix (Figure 6-30F), followed by DNA ligation (Figure 6-30G). 

double-strand break 

daughter DNA 










Figure 6-30 Homologous recombination 
allows the flawless repair of DNA double- 
strand breaks. This is the preferred method 
for repairing double-strand breaks that arise 
shortly after the DNA has been replicated 
but before the cell has divided. See text 
for details. (Adapted from M. McVey et al., 
Proc. Nati. Acad. Sci. USA 101:15694-15699, 
2004. With permission from the National 
Academy of Sciences.) 

LibertadDigital \ 2015 



DNA Replication, Repair, and Recombination 

single DNA strand of 
normal P-globin gene 



Figure 6-31 A single nucleotide change 
causes the disease sickle-cell anemia. 

(A) |3-glob¡n ¡s one of the two types of 
proteln subunlts that form hemoglobin (see 
Figure 4-24). A single nucleotide change 
(mutation) in the P-globin gene produces 
a P-globin subunit that differs from normal 
P-globin only by a change from glutamlc 
acid to valine at the sixth amino acid 
position. (Only a small portion of the gene 
¡s shown here; the P-globin subunit contains 
a total of 146 amino acids.) Flumans carry 
two copies of each gene (one inherited from 
each parent); a sickle-cell mutation in one of 
the two P-globin genes generally causes no 
harm to the individual, as it is compensated 
for by the normal gene. Flowever, an 
individual who inherits two copies of the 
mutant p-globin gene will have sickle-cell 
anemia. Normal red blood cells are shown 
in (B), and those from an individual suffering 
from sickle-cell anemia in (C). Although 
sickle-cell anemia can be a life-threatening 
disease, the mutation responsible can also 
be beneficial. People with the disease, 
or those who carry one normal gene and 
one sickle-cell gene, are more resistant to 
malaria than unaffected individuáis, because 
the parasite that causes malaria grows 
poorly in red blood cells that contain the 
sickle-cell form of hemoglobin. 

The net result is two intact DNA hélices, where the genetic information 
from one was used as a témplate to repair the other. 

Homologous recombination can also be used to repair many other types 
of DNA damage, making it perhaps the most handy DNA repair mech- 
anism available to the cell: all that is needed is an intact homologous 
chromosome to use as a partner—a situation that occurs transiently each 
time a chromosome is duplicated. The "all-purpose" nature of homolo¬ 
gous recombinational repair probably explains why this mechanism, and 
the proteins that carry it out, have been conserved in virtually all cells on 

Homologous recombination is versatile, and has a crucial role in the 
exchange of genetic information during the formation of the germ cells— 
sperm and eggs. This specialized process, called meiosis, enhances the 
generation of genetic diversity within a species during sexual reproduc- 
tion. We will discuss it when we talk about sex in Chapter 19. 

Failure to Repair DNA Damage Can Have Severe 
Consequences for a Cell or Organism 

On occasion, the cell’s DNA replication and repair processes fail and give 
rise to a mutation. This permanent change in the DNA sequence can have 
profound consequences. A mutation that affects just a single nucleotide 
pair can severely compromise an organism's fitness if the change occurs 
in a vital position in the DNA sequence. Because the structure and activ- 
ity of each protein depend on its amino acid sequence, a protein with an 
altered sequence may function poorly or not at all. For example, humans 
use the protein hemoglobin to transport oxygen in the blood (see Figure 
4-24). A permanent change in a single nucleotide in a hemoglobin gene 
can cause cells to make hemoglobin with an incorrect sequence of amino 
acids. One such mutation causes the disease sickle-cell anemia. The 
sickle-cell hemoglobin is less soluble than normal hemoglobin and forms 
fibrous intracellular precipitates, which produce the characteristic sickle 
shape of affected red blood cells (Figure 6-31). Because these cells are 
more fragüe and frequently tear as they travel through the bloodstream, 
patients with this potentially life-threatening disease have fewer red blood 
cells than usual—that is, they are anemic. This anemia can cause weak- 
ness, dizziness, headaches, and breathlessness. Moreover, the abnormal 
red blood cells can aggregate and block small vessels, causing pain and 
organ failure. We know about sickle-cell hemoglobin because individu¬ 
áis with the mutation survive; the mutation even provides a benefit—an 
increased resistance to malaria. Over the course of evolution, many other 
mutations in the hemoglobin gene have arisen, but only those that do not 
completely destroy the protein remain in the population. 

The example of sickle-cell anemia, which is an inherited disease, illus- 
trates the importance of protecting reproductive cells {germ cells) against 
mutation. A mutation in a germ cell will be passed on to all the cells in 
the body of the multicellular organism that develops from it, including the 
germ cells responsible for the production of the next generation. 

The many other cells in a multicellular organism (its somatic cells) must 
also be protected against mutation—in this case, against mutations that 
arise during the life of an individual. Nucleotide changes that occur in 
somatic cells can give rise to variant cells, some of which grow and 
divide in an uncontrolled fashion at the expense of the other cells in the 
organism. In the extreme case, an unchecked cell proliferation known 
as cáncer results. Cancers are responsible for about 30% of the deaths 
that occur in Europe and North America, and they are caused largely by 
a gradual accumulation of random mutations in a somatic cell and its 

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DNA Repair 


Figure 6-32 Cáncer incidence increases dramatically with age. The 

number of newly diagnosed cases of cáncer of the colon ¡n women 
¡n England and Wales in one year ¡s plotted as a function of age at 
diagnosis. Colon cáncer, like most human cancers, is caused by the 
accumulation of múltiple mutations. Because cells are continually 
experienclng accidental changes to their DNA—which accumulate and 
are passed on to progeny cells when the mutated cells divide—the 
chance that a cell will become cancerous ¡ncreases greatly with age. 
(Data from C. Muir et al.. Cáncer Incidence in Five Continents, Vol. V. 
Lyon: International Agency for Research on Cáncer, 1987.) 

descendants (Figure 6-32). Increasing the mutation frequency even two- 
or threefold could cause a disastrous increase in the incidence of cáncer 
by accelerating the rate at which such somatic cell variants arise. 

Thus, the high fidelity with which DNA sequences are replicated and 
maintained is important both for reproductive cells, which transmit the 
genes to the next generation, and for somatic cells, which normally func¬ 
tion as carefully regulated members of the complex community of cells 
in a multicellular organism. We should therefore not be surprised to find 
that all cells possess a veiy sophisticated set of mechanisms to reduce 
the number of mutations that occur in their DNA, devoting hundreds of 
genes to these repair processes. 



A Record of the Fidelity of DNA Replication and Repair Is 
Preserved in Genome Sequences 

Although the majority of mutations do neither harm ñor good to an 
organism, those that have harmful consequences are usually eliminated 
from the population through natural selection; individuáis cariying the 
altered DNA may die or experience decreased fertility, in which case 
these changes will be lost. By contrast, favorable changes will tend to 
persist and spread. 

But even where no selection operates—at the many sites in the DNA 
where a change of nucleotide has no effect on the fitness of the organ¬ 
ism—the genetic message has been faithfully preserved over tens of 
millions of years. Thus humans and chimpanzees, after about 5 million 
years of divergent evolution, still have DNA sequences that are at least 
98% identical. Even humans and whales, after 10 or 20 times this amount 
of time, have chromosomes that are unmistakably similar in their DNA 
sequence, and many proteins have amino acid sequences that are almost 
identical (Figure 6-33). Thus our genome—and those of our relatives— 
contains a message from the distant past. Thanks to the faithfulness of 
DNA replication and repair, 100 million years of evolution have scarcely 
changed its essential content. 



11111111111 Mili IIIII MI 1111IIII 





Figure 6-33 The sex-determination 
genes from humans and whales are 
unmistakably similar. Although their body 
plans are strikingly dlfferent, humans and 
whales are bullt from the same proteins. 
Desplte the many millions of years that 
have passed since humans and whales 
diverged, the nucleotide sequences of 
many of their genes are closely similar. The 
DNA sequences of a part of the gene that 
determines maleness in humans and in 
whales are shown, one above the other; the 
positions where the two are identical are 
shaded in green. 

LibertadDigital \ 2015 

220 CHAPTER 6 DNA Replication, Repair, and Recombination 


• Before a cell divides, it must accurately replícate the vast quantity of 
genetic information carried in its DNA. 

• Because the two strands of a DNA double helix are complementary, 
each strand can act as a témplate for the synthesis of the other. Thus 
DNA replication produces two identical, double-helical DNA mole- 
cules, enabling genetic information to be copied and passed on from 
a cell to its daughter cells and from a parent to its offspring. 

• During replication, the two strands of a DNA double helix are pulled 
apart at a replication origin to form two Y-shaped replication forks. 
DNA polymerases at each fork produce a new complementary DNA 
strand on each parental strand. 

• DNA polymerase replicates a DNA témplate with remarkable fidel- 
ity, making only about one error in eveiy 10 7 nucleotides copied. 
This accuracy is made possible, in part, by a proofreading process in 
which the enzyme corrects its own mistakes as it moves along the 

• Because DNA polymerase synthesizes new DNA in only one direction, 
only the leading strand at the replication fork can be synthesized in 
a continuous fashion. On the lagging strand, DNA is synthesized in 
a discontinuous backstitching process, producing short fragments of 
DNA that are later joined together by DNA ligase. 

• DNA polymerase is incapable of starting a new DNA chain from 
scratch. Instead, DNA synthesis is primed by an RNA polymerase 
called primase, which makes short lengths of RNA primers that are 
then elongated by DNA polymerase. These primers are subsequently 
erased and replaced with DNA. 

• DNA replication requires the cooperation of many proteins that form 
a multienzyme replication machine that copies both DNA strands as 
it moves along the double helix. 

• In eukaiyotes, a special enzyme called telomerase replicates the 
DNA at the ends of the chromosomes. 

• The rare copying mistakes that escape proofreading are dealt with by 
mismatch repair proteins, which increase the accuracy of DNA repli¬ 
cation to one mistake per 10 9 nucleotides copied. 

• Damage to one of the two DNA strands, caused by unavoidable 
Chemical reactions, is repaired by a variety of DNA repair enzymes 
that recognize damaged DNA and excise a short stretch of the dam- 
aged strand. The missing DNA is then resynthesized by a repair DNA 
polymerase, using the undamaged strand as a témplate. 

• If both DNA strands are broken, the double-strand break can be rap- 
idly repaired by nonhomologous end joining. Nucleotides are lost in 
the process, altering the DNA sequence at the repair site. 

• Homologous recombination can flawlessly repair double-strand 
breaks using an undamaged homologous double helix as a témplate. 

• Highly accurate DNA replication and DNA repair processes play a key 
role in protecting us from the uncontrolled growth of somatic cells 
known as cáncer. 

LibertadDigital \ 2015 

Chapter 6 End-of-Chapter Questions 




DNA ligase 

DNA polymerase 

DNA repair 

DNA replication 

homologous recombination 

lagging strand 

leading strand 

mismatch repair 


nonhomologous end joining 

Okazaki fragment 



replication fork 

replication origin 

RNA (ribonucleic acid) 






DNA mismatch repair enzymes preferentially repair bases 
on the newly synthesized DNA strand, using the oíd DNA 
strand as a témplate. If mismatches were simply repaired 
without regard for which strand served as témplate, would 
this reduce replication errors? Explain your answer. 


Suppose a mutation affects an enzyme that is required to 
repair the damage to DNA caused by the loss of purine 
bases. The loss of a purine occurs about 5000 times in the 
DNA of each of your cells per day. As the average difference 
in DNA sequence between humans and chimpanzees is 
about 1%, how long will it take you to turn into an ape? 
What is wrong with this argument? 


Which of the following statements are corred? Explain your 

A. A baderial replication fork is asymmetrical because 
it contains two DNA polymerase molecules that are 
strudurally distind. 

B. Okazaki fragments are removed by a nuclease that 
degrades RNA. 

C. The error rate of DNA replication is reduced both by 
proofreading by DNA polymerase and by DNA mismatch 

D. In the absence of DNA repair, genes are unstable. 

E. None of the aberrant bases formed by deamination 
occur naturally in DNA. 

F. Cáncer can result from the accumulation of mutations in 
somatic cells. 


The speed of DNA replication at a replication fork is about 
100 nucleotides per second in human cells. What is the 
mínimum number of origins of replication that a human cell 
must have if it is to replícate its DNA once every 24 hours? 
Recall that a human cell contains two copies of the human 
genome, one inherited from the mother, the other from the 
father, each consisting of 3 X 10 9 nucleotide pairs. 


Look carefully at Figure 6-11 and at the strudures of the 
compounds shown in Figure Q6-9. 

Figure Q6-9 

A. What would you exped if ddCTP were added to a DNA 
replication readion in large excess over the concentration of 
the available deoxycytosine triphosphate (dCTP), the normal 
deoxycytosine triphosphate? 

B. What would happen if it were added at 10% of the 
concentration of the available dCTP? 

C. What effeds would you exped if ddCMP were added 
under the same conditions? 

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CHAPTER 6 DNA Replication, Repair, and Recombination 


Figure Q6-10 shows a snapshot of a replication fork in 
which the RNA primer has just been added to the lagging 
strand. Using this diagram as a guide, sketch the path of the 
DNA as the next Okazaki fragment is synthesized. Indícate 
the sliding clamp and the single-strand DNA-binding protein 
as appropriate. 

Approximately how many high-energy bonds does DNA 
polymerase use to replícate a bacterial chromosome 
(¡gnoring helicase and other enzymes associated with the 
replication fork)? Compared with its own dry weight of 
10 -12 g, how much glucose does a single bacterium need to 
provide enough energy to copy its DNA once? The number 
of nucleotide pairs in the bacterial chromosome is 3 X 10 6 . 
Oxidation of one glucose molecule yields about 30 high- 
energy phosphate bonds. The molecular weight of glucose 
is 180 g/mole. (Recall from Figure 2-3 that a mole consists 
of 6 X 10 23 molecules.) 


Describe the consequences that would arise if a eukaryotic 

A. Contained only one origin of replication: 

(i) at the exact center of the chromosome 

(ii) at one end of the chromosome 

B. Lacked one or both telomeres 

C. Had no centromere 

Assume that the chromosome is 150 million nucleotide pairs 
in length, a typical size for an animal chromosome, and 
that DNA replication in animal cells proceeds at about 100 
nucleotides per second. 


Because DNA polymerase proceeds only in the 5'-to-3' 
direction, the enzyme is able to corred its own 
polymerization errors as it moves along the DNA (Figure 
Q6-16). A hypothetical DNA polymerase that synthesized 
in the 3'-to-5' diredion would be unable to proofread. 

Given what you know about nucleic acid chemistry and DNA 
synthesis, draw a sketch similar to Figure Q6-16 that shows 
what would happen if a DNA polymerase operating in the 
3'-to-5' diredion were to remove an incorred nucleotide 
from a growing DNA strand. Why would the edited strand 
be unable to be elongated? 


5' 3' 

_end of growing 

DNA strand 

■ ■ 


What, if anything, is wrong with the following statement: 
"DNA stability in both reprodudive cells and somatic cells is 
essential for the survival of a species." Explain your answer. 


A common type of Chemical 
damage to DNA is produced l ' 

by a spontaneous readion 1 | 1 

termed deamination, in which T 

a nucleotide base loses an 

amino group (NH2). The amino Figure Q6-13 

group is replaced by a keto group (C=0), by the general 
readion shown in Figure Q6-13. Write the strudures of the 
bases A, G, C, T, and U and predid the produds that will 
be produced by deamination. By looking at the produds of 
this readion—and remembering that, in the cell, these will 
need to be recognized and repaired—can you propose an 
explanation for why DNA cannot contain uracil? 


A. Explain why telomeres and telomerase are needed 
for replication of eukaryotic chromosomes but not for 
replication of a circular baderial chromosome. Draw a 
diagram to ¡Ilústrate your explanation. 

B. Would you still need telomeres and telomerase to 
complete eukaryotic chromosome replication if primase 
always laid down the RNA primer at the very 3' end of the 
témplate for the lagging strand? 






LibertadDigital \ 2015 


From DNA to Protein: 

How Cells Read the Genome 

Once the double-helical structure of DNA (deoxyribonucleic acid) had 
been determined in the early 1950s, it became clear that the hereditaiy 
information in cells is encoded in the linear order—or sequence —of the 
four different nucleotide subunits that make up the DNA. We saw in 
Chapter 6 how this information can be passed on unchanged from a cell 
to its descendants through the process of DNA replication. But how does 
the cell decode and use the information? How do genetic instructions 
written in an alphabet of just four "letters" direct the formation of a bac- 
terium, a fruit fly, or a human? We still have a lot to learn about how the 
information stored in an organism's genes produces even the simplest 
unicellular bacterium, let alone how it directs the development of com- 
plex multicellular organisms like ourselves. But the DNA code itself has 
been deciphered, and we have come a long way in understanding how 
cells read it. 

Even before the DNA code was broken, it was known that the information 
contained in genes somehow directed the synthesis of proteins. Proteins 
are the principal constituents of cells and determine not only cell struc¬ 
ture but also cell function. In previous chapters, we encountered some 
of the thousands of different kinds of proteins that cells can make. We 
saw in Chapter 4 that the properties and function of a protein molecule 
are determined by the sequence of the 20 different amino acid subunits 
in its polypeptide chain: each type of protein has its own unique amino 
acid sequence, which dictates how the chain will fold to form a molecule 
with a distinctive shape and chemistry. The genetic instructions carried 
by DNA must therefore specify the amino acid sequences of proteins. We 
will see in this chapter exactly how this is done. 




LibertadDigital \ 2015 


CHAPTER 7 From DNAto Protein: How Cells Read the Genome 

( DNA 




amino acids 

Figure 7-1 Genetic ¡nformation directs 
the synthesis of proteins. The flow of 
genetic ¡nformation from DNAto RNA 
(transcription) and from RNA to protein 
(translation) occurs in all living cells. It 
was Francis Crick who dubbed this flow 
of ¡nformation "the central dogma." The 
segments of DNA that are transcribed into 
RNA are called genes. 

DNA does not synthesize proteins itself, but it acts like a manager, del- 
egating the various tasks to a team of workers. When a particular protein 
is needed by the cell, the nucleotide sequence of the appropriate segment 
of a DNA molecule is first copied into another type of nucleic acid —RNA 
(ribonucleic acid). That segment of DNA is called a gene, and the result- 
ing RNA copies are then used to direct the synthesis of the protein. Many 
thousands of these conversions from DNA to protein occur eveiy sec- 
ond in each cell in our body. The flow of genetic ¡nformation in cells is 
therefore from DNA to RNA to protein (Figure 7-1 ). All cells, from bacte¬ 
ria to humans, express their genetic information in this way—a principie 
so fundamental that it has been termed the central dogma of molecular 

In this chapter, we explain the mechanisms by which cells copy DNA 
into RNA (a process called transcription) and then use the information 
in RNA to make protein (a process called translation). We also discuss 
a few of the key variations on this basic scheme. Principal among these 
is RNA splicing, a process in eukaiyotic cells in which segments of an 
RNA transcript are removed—and the remaining segments stitched back 
together—before the RNA is translated into protein. In the final section, 
we consider how the present scheme of information storage, transcrip¬ 
tion, and translation might have arisen from much simpler Systems in the 
earliest stages of cell evolution. 


Transcription and translation are the means by which cells read out, or 
express, the instructions in their genes. Many identical RNA copies can be 
made from the same gene, and each RNA molecule can direct the syn¬ 
thesis of many identical protein molecules. This successive amplification 
enables cells to rapidly synthesize large amounts of protein whenever 
necessaiy. At the same time, each gene can be transcribed, and its RNA 
translated, at different rates, providing the cell with a way to make vast 
quantities of some proteins and tiny quantities of others (Figure 7-2). 
Moreover, as we discuss in Chapter 8, a cell can change (or regúlate) the 
expression of each of its genes according to the needs of the moment. 
In this section, we discuss the production of RNA, the first step in gene 


Consider the expression "central 
dogma," which refers to the flow 
of genetic ¡nformation from DNA 
to RNA to protein. Is the word 
"dogma" appropriate in this 

Figure 7-2 A cell can express different 
genes at different rates. In this and later 
figures, the untranscribed portions of the 
DNA are shown in gray. 



A A. A A A 

A A A A A 

A A A A A 

A A A A A 

• # # 9 $ 







LibertadDigital \ 2015 

From DNA to RNA 



OLI I? OI 1 



used in RNA 



used in DNA 


sed in RNA 

Figure 7-3 The Chemical structure of RNA differs slightly from 
that of DNA. (A) RNA contains the sugar ribose, which differs from 
deoxyribose, the sugar used in DNA, by the presence of an additional 
-OH group. (B) RNA contains the base uracil, which differs from 
thymine, the equivalent base in DNA, by the absence of a -CH3 group. 
(C) A short length of RNA. The Chemical linkage between nucleotides 
in RNA—a phosphodiester bond—¡s the same as that in DNA. 

Portions of DNA Sequence Are Transcribed ¡nto RNA 

The first step a cell takes in expressing one of its many thousands of 
genes is to copy the nucleotide sequence of that gene into RNA. The proc- 
ess is called transcription because the information, though copied into 
another Chemical form, is still written in essentially the same language— 
the language of nucleotides. Like DNA, RNA is a linear polymer made 
of four different nucleotide subunits, linked together by phosphodiester 
bonds. It differs from DNA chemically in two respects: (1) the nucle¬ 
otides in RNA are ribonucleotides —that is, they contain the sugar ribose 
(henee the ñame ribonucleic acid) rather than deoxyribose; (2) although, 
like DNA, RNA contains the bases adenine (A), guanine (G), and cyto- 
sine (C), it contains uracil (U) instead of the thymine (T) found in DNA 
(Figure 7-3). Because U, like T, can base-pair by hydrogen-bonding with 
A (Figure 7-4), the complementaiy base-pairing properties described for 
DNA in Chapter 5 apply also to RNA. 

Although their Chemical differences are small, DNA and RNA differ quite 
dramatically in overall structure. Whereas DNA always occurs in cells 
as a double-stranded helix, RNA is single-stranded. This difference has 
important functional consequences. Because an RNA chain is single- 
stranded, it can fold up into a variety of shapes, just as a polypeptide 
chain folds up to form the final shape of a protein (Figure 7-5); double- 
stranded DNA cannot fold in this fashion. As we discuss later, the ability 
to fold into a complex three-dimensional shape allows RNA to cariy out 
various functions in cells, in addition to conveying information between 
DNA and protein. Whereas DNA functions solely as an information store, 
some RNAs have structural, regulatoiy, or catalytic roles. 

Figure 7-4 Uracil forms a base pair with adenine. The hydrogen 
bonds that hold the base pair together are shown in red. Uracil has the 
same base-pairing properties as thymine. Thus U-A base pairs in RNA 
closely resemble T-A base pairs in DNA (see Figure 5-6A). 

5' end 

I U | uracil 

sugar-phosphate backbone 

LibertadDigital \ 2015 


CHAPTER 7 From DNAto Protein: How Cells Read the Genome 

Figure 7-5 RNA molecules can form intramolecular base pairs and fold into specific structures. RNA ¡s single- 
stranded, but ¡t often contains short stretches of nucleotides that can base-pair with complementary sequences 
found elsewhere on the same molecule. These interactions—along with some "nonconventional base-pair 
¡nteractions (e.g., A-G)—allow an RNA molecule to fold into a three-dimensional structure that is determined by its 
sequence of nucleotides. (A) A diagram of a hypothetical, folded RNA structure showing only conventional (G-C and 
A-U) base-pair ¡nteractions. (B) Incorporating nonconventional base-pair ¡nteractions (greerí) changes the structure of 
the hypothetical RNA shown ¡n (A). (C) Structure of an actual RNA molecule that is ¡nvolved ¡n RNA splicing. This RNA 
contains a considerable amount of double-helical structure. The sugar-phosphate backbone is blue and the bases are 
red; the conventional base-pair interactions are indicated by red "rungs" that are continuous, and nonconventional 
base pairs are indicated by broken red rungs. For an additional view of RNA structure, see Movie 7.1. 

coding strand DNA 

Figure 7-6 Transcription of a gene 
produces an RNA complementary to one 
strand of DNA. The transcribed strand 
of the gene, the bottom strand ¡n this 
example, is called the témplate strand. 

The nontemplate strand of the gene 
(here, shown at the top) is sometimes called 
the coding strand because its sequence 
is equivalent to the RNA product, as 
shown. Which DNA strand serves as the 
témplate varíes, depending on the gene, 
as we discuss later. By convention, an RNA 
molecule is always depicted with its 
5' end—the first part to be synthesized— 
to the left. 

Transcription Produces RNA That Is Complementary to 
One Strand of DNA 

All the RNA in a cell is made by transcription, a process that has certain 
similarities to DNA replication (discussed in Chapter 6). Transcription 
begins with the opening and unwinding of a small portion of the DNA 
double helix to expose the bases on each DNA strand. One of the two 
strands of the DNA double helix then acts as a témplate for the synthe- 
sis of RNA. Ribonucleotides are added, one by one, to the growing RNA 
chain; as in DNA replication, the nucleotide sequence of the RNA chain 
is determined by complementary base-pairing with the DNA témplate. 
When a good match is made, the incoming ribonucleotide is covalently 
linked to the growing RNA chain by the enzyme RNA polymemse. The 
RNA chain produced by transcription—the RNA transcript —is therefore 
elongated one nucleotide at a time and has a nucleotide sequence exactly 
complementary to the strand of DNA used as the témplate (Figure 7-6). 
Transcription differs from DNA replication in several crucial respects. 
Unlike a newly formed DNA strand, the RNA strand does not remain 
hydrogen-bonded to the DNA témplate strand. Instead, just behind the 
región where the ribonucleotides are being added, the RNA chain is dis¬ 
placed and the DNA helix re-forms. For this reason—and because only 
one strand of the DNA molecule is transcribed—RNA molecules are 
single-stranded. Further, because RNAs are copied from only a limited 
región of DNA, RNA molecules are much shorter than DNA molecules; 
DNA molecules in a human chromosome can be up to 250 million nucle¬ 
otide pairs long, whereas most mature RNAs are no more than a few 
thousand nucleotides long, and many are much shorter than that. 

LibertadDigital \ 2015 

From DNA to RNA 


RNA polymerase 

newly synthesized polymerase triphosphate 

RNAtranscript tunnel 

Like the DNA polymerase that carries out DNA replication (discussed 
in Chapter 6), RNA polymerases catalyze the formation of the phos- 
phodiester bonds that link the nucleotides together and form the 
sugar-phosphate backbone of the RNA chain (see Figure 7-3). The RNA 
polymerase moves stepwise along the DNA, unwinding the DNA helix 
just ahead to expose a new región of the témplate strand for comple- 
mentaiy base-pairing. In this way, the growing RNA chain is extended by 
one nucleotide at a time in the 5'-to-3' direction (Figure 7-7). The incom- 
ing ribonucleoside triphosphates (ATP, CTP, UTP, and GTP) provide the 
energy needed to drive the reaction forward (see Figure 6-11). 

The almost immediate release of the RNA strand from the DNA as it is syn¬ 
thesized means that many RNA copies can be made from the same gene 
in a relatively short time; the synthesis of the next RNA is usually started 
before the flrst RNA has been completed (Figure 7-8). A medium-sized 
gene—say, 1500 nucleotide pairs—requires approximately 50 seconds for 
a molecule of RNA polymerase to transcribe it (Movie 7.2). At any given 
time, there could be dozens of polymerases speeding along this single 
stretch of DNA, hard on one another's heels, allowing more than 1000 
transcripts to be synthesized in an hour. For most genes, however, the 
amount of transcription is much less than this. 

Although RNA polymerase catalyzes essentially the same Chemical reac¬ 
tion as DNA polymerase, there are some important differences between 
the two enzymes. First, and most obviously, RNA polymerase uses ribo¬ 
nucleoside for phosphates as substrates, so it catalyzes the linkage of 
ribonucleotides, not deoxyribonucleotides. Second, unlike the DNA 
polymerase involved in DNA replication, RNA polymerases can start an 
RNA chain without a primer. This difference likely evolved because tran¬ 
scription need not be as accurate as DNA replication; unlike DNA, RNA is 
not used as the permanent storage form of genetic information in cells, 
so mistakes in RNA transcripts have relatively minor consequences for a 
cell. RNA polymerases make about one mistake for every 10 4 nucleotides 
copied into RNA, whereas DNA polymerase makes only one mistake for 
every 10 7 nucleotides copied. 

Cells Produce Various Types of RNA 

The vast majority of genes carried in a cell's DNA specify the amino acid 
sequences ofproteins. The RNAmolecules encodedby these genes—which 

Figure 7-7 DNA is transcribed into RNA 
by the enzyme RNA polymerase. RNA 

polymerase (pa/e blué) moves stepwise 
along the DNA, unwinding the DNA helix in 
front of it. As it progresses, the polymerase 
adds ribonucleotides one by one to the 
RNA chain, using an exposed DNA strand as 
a témplate. The resulting RNAtranscript is 
thus single-stranded and complementary to 
this témplate strand (see Figure 7-6). As the 
polymerase moves along the DNA témplate 
(in the 3'-to-5' direction), it displaces the 
newly formed RNA, allowing the two strands 
of DNA behind the polymerase to rewind. 

A short región of hybrid DNA/RNA helix 
(approximately nine nucleotides in length) 
therefore forms only transiently, causing a 
"window" of DNA/RNA helix to move along 
the DNA with the polymerase (Movie 7.2). 


In the electrón micrograph in Figure 
7-8, are the RNA polymerase 
molecules moving from right to left 
or from left to right? Why are the 
RNA transcripts so much shorter 
than the DNA segments (genes) that 
encode them? 

Figure 7-8 Transcription can be visualized 
in the electrón microscope. The 

micrograph shows many molecules of RNA 
polymerase simultaneously transcribing two 
adjacent ribosomal genes on a single DNA 
molecule. Molecules of RNA polymerase 
are barely visible as a series of tiny dots 
along the spine of the DNA molecule; 
each polymerase has an RNA transcript (a 
short, fine thread) radiating from it. The 
RNA molecules being transcribed from the 
two ribosomal genes—ribosomal RNAs 
(rRNAs)—are not translated into protein, but 
are instead used directly as components of 
ribosomes, macromolecular machines made 
of RNA and protein. The large partióles that 
can be seen at the free, 5' end of each rRNA 
transcript are believed to be ribosomal 
proteins that have assembled on the ends 
of the growing transcripts. (Courtesy of 
Ulrich Scheer.) 

LibertadDigital \ 2015 

228 CHAPTER 7 From DNA to Protein: How Cells Read the Genome 

ultimately direct the synthesis of proteins—are called messenger RNAs 
(mRNAs). In eukaryotes, each mRNA typically carries information tran- 
scribed from just one gene, which codes for a single protein; in bacteria, a 
set of adjacent genes is often transcribed as a single mRNA, which there- 
fore carries the information for several different proteins. 

The final product of other genes, however, is the RNA itself. As we see 
later, these nonmessenger RNAs, like proteins, have various roles, serv- 
ing as regulatory, structural, and catalytic components of cells. They play 
key parts, for example, in translating the genetic message into protein: 
ribosomal RNAs (rRNAs) form the structural and catalytic core of the ribos- 
ornes, which transíate mRNAs into protein, and transfer RNAs (tRNAs) act 
as adaptors that select specific amino acids and hold them in place on a 
ribosome for their incorporation into protein. Other small RNAs, called 
microRNAs (miRNAs ), serve as key regulators of eukaiyotic gene expres- 
sion, as we discuss in Chapter 8. The most common types of RNA are 
summarized in Table 7-1 . 

In the broadest sense, the term gene expression refers to the process 
by which the information encoded in a DNA sequence is translated into 
a product that has some effect on a cell or organism. In cases where 
the final product of the gene is a protein, gene expression ineludes both 
transcription and translation. When an RNA molecule is the gene's final 
product, however, gene expression does not require translation. 

Signáis in DNA Tell RNA Polymerase Where to Start and 
Finish Transcription 

The initiation of transcription is an especially critical process because it 
is the main point at which the cell seleets which proteins or RNAs are to 
be produced. To begin transcription, RNA polymerase must be able to 
recognize the start of a gene and bind firmly to the DNA at this site. The 
way in which RNA polymerases recognize the transcription start site of a 
gene differs somewhat between bacteria and eukaryotes. Because the 
situation in bacteria is simpler, we describe it first. 

When an RNA polymerase collides randomly with a DNA molecule, the 
enzyme sticks weakly to the double helix and then slides rapidly along its 
length. RNA polymerase latches on tightly only after it has encountered 
a gene región called a promoter, which contains a specific sequence 
of nucleotides that lies immediately upstream of the starting point for 
RNA synthesis. Once bound tightly to this sequence, the RNA polymerase 
opens up the double helix immediately in front of the promoter to expose 
the nucleotides on each strand of a short stretch of DNA. One of the two 
exposed DNA strands then acts as a témplate for complementary base- 
pairing with incoming ribonucleoside triphosphates, two of which are 


1 Type of RNA 

Function ! 

messenger RNAs (mRNAs) 

code for proteins 

ribosomal RNAs (rRNAs) 

form the core of the ribosome's structure and 
catalyze protein synthesis 

microRNAs (miRNAs) 

regúlate gene expression 

transfer RNAs (tRNAs) 

serve as adaptors between mRNA and amino acids 
during protein synthesis 

other noncoding RNAs 

used in RNA splicing, gene regulation, telomere 
maintenance, and many other processes 

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From DNA to RNA 


Figure 7-9 Signáis in the nudeotide 
sequence of a gene tell bacterial RNA 
polymerase where to start and stop 
transcription. Bacterial RNA polymerase 
(light blue ) contains a subunit called sigma 
factor (yellow) that recognizes the promoter 
of a gene (greerí). Once transcription has 
begun, sigma factor is released, and the 
polymerase moves forward and continúes 
synthesizing the RNA. Chain elongation 
continúes until the polymerase encounters a 
sequence in the gene called the terminator 
(red). There the enzyme halts and releases 
both the DNA témplate and the newly 
made RNA transcript. The polymerase then 
reassociates with a free sigma factor and 
searches for another promoter to begin the 
process again. 

joined together by the polymerase to begin synthesis of the RNA chain. 
Chain elongation then continúes until the enzyme encounters a second 
signal in the DNA, the terminator (or stop site), where the polymerase 
halts and releases both the DNA témplate and the newly made RNA tran¬ 
script (Figure 7-9). This terminator sequence is contained within the 
gene and is transcribed into the 3' end of the newly made RNA. 

Because the polymerase must bind tightly before transcription can begin, 
a segment of DNA will be transcribed only if it is preceded by a promoter. 
This ensures that those portions of a DNA molecule that contain a gene 
will be transcribed into RNA. The nucleotide sequences of a typical pro¬ 
moter—and a typical terminator—are presented in Figure 7-10. 

In bacteria, it is a subunit of RNA polymerase, the sigma (a) factor (see 
Figure 7-9), that is primarily responsible for recognizing the promoter 
sequence on the DNA. But how can this factor "see" the promoter, given 
that the base-pairs in question are situated in the interior of the DNA 
double helix? It tums out that each base presents unique features to the 
outside of the double helix, allowing the sigma factor to find the promoter 
sequence without having to sepárate the entwined DNA strands. 

The next problem an RNA polymerase faces is determining which of 
the two DNA strands to use as a témplate for transcription: each strand 
has a different nucleotide sequence and would produce a different RNA 
transcript. The secret lies in the structure of the promoter itself. Eveiy 
promoter has a certain polarity: it contains two different nucleotide 
sequences upstream of the transcriptional start site that position the RNA 
polymerase, ensuring that it binds to the promoter in only one orientation 

LibertadDigital \ 2015 


CHAPTER 7 From DNAto Protein: How Cells Read the Genome 

Figure 7-10 Bacterial promoters 
and terminators have specific 
nudeotide sequences that are 
recognized by RNA polymerase. 

(A) The green-shaded regions 
represent the nudeotide sequences 
that specify a promoter. The 
numbers above the DNA indícate 
the positions of nucleotides 
counting from the first nudeotide 
transcribed, which ¡s designated +1. 
The polarity of the promoter orients 
the polymerase and determines 
which DNA strand ¡s transcribed. 

All bacterial promoters contain 
DNA sequences at -10 and -35 
that closely resemble those shown 
here. (B) The red-shaded regions 
represent sequences in the gene 
that signal the RNA polymerase to 
termínate transcription. Note that 
the regions transcribed into RNA 
contain the terminator but not the 
promoter nudeotide sequences. By 
convention, the sequence of a gene 
¡s that of the non-template strand, as 
this strand has the same sequence 
as the transcribed RNA (with T 
substituting for U). 

(A) PROMOTER I -35 -10 



3' - atcacat^^^Hactatcttcgtgagatgatataag 

i ^ témplate strand 




3' - gggtgt^^^^^Baaggc^^^^^Bt^^Bttgaaagaaattact- 5’ J 


témplate strand I s ¡ te 

5' ■ 3' RNA 

(see Figure 7-10A). Because the polymerase can only synthesize RNA 
in the 5'-to-3' direction once the enzyme is bound it must use the DNA 
strand oriented in the 3'-to-5' direction as its témplate. 

This selection of a témplate strand does not mean that on a given chro- 
mosome, transcription always proceeds in the same direction. With 
respect to the chromosome as a whole, the direction of transcription var¬ 
íes from gene to gene. But because each gene typically has only one 
promoter, the orientation of its promoter determines in which direction 
that gene is transcribed and therefore which strand is the témplate strand 
(Figure 7-11). 

Figure 7-11 On an individual 
chromosome, some genes are transcribed 
using one DNA strand as a témplate, 
and others are transcribed from the 
other DNA strand. RNA polymerase 
always moves in the 3'-to-5' direction 
and the selection of the témplate strand 
is determined by the orientation of the 
promoter (green arrowheads) at the 
beginning of each gene. Thus the genes 
transcribed from left to right use the bottom 
DNA strand as the témplate (see Figure 
7-10); those transcribed from right to left 
use the top strand as the témplate. 

Initiation of Eukaryotic Gene Transcription Is a Complex 

Many of the principies we just outlined for bacterial transcription also 
apply to eukaryotes. However, transcription initiation in eukaryotes dif- 
fers in several important ways from that in bacteria: 

• The first difference lies in the RNA polymerases themselves. While 
bacteria contain a single type of RNA polymerase, eukaryotic cells 
have three— RNA polymerase I, RNA polymerase II, and RNA polymer¬ 
ase III. These polymerases are responsible for transcribing different 
types of genes. RNA polymerases I and III transcribe the genes encod- 
ing transfer RNA, ribosomal RNA, and various other RNAs that play 
structural and catalytic roles in the cell (Table 7-2). RNA polymerase 
II transcribes the vast majority of eukaryotic genes, including all those 
that encode proteins and miRNAs (Movie 7.3). Our subsequent dis- 
cussion will therefore focus on RNA polymerase II. 

• A second difference is that, whereas the bacterial RNA polymerase 
(along with its sigma subunit) is able to initiate transcription on its 
own, eukaryotic RNA polymerases require the assistance of a large set 
of accessory proteins. Principal among these are the general transcrip¬ 
tion factors, which must assemble at each promoter, along with the 
polymerase, before the polymerase can begin transcription. 

RNA transcript 
from gene b 


|—I gene a 

RNA transcript 
from gene a 

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From DNA to RNA 



1 Type of Polymerase 1 

Genes Transcribed I 

RNA polymerase 1 

most rRNA genes 

RNA polymerase II 

all protein-coding genes, miRNA genes, plus 
genes for other noncoding RNAs (e.g., those in 

RNA polymerase III 

tRNA genes 

5S rRNA gene 

genes for many other small RNAs 

• A third distinctive feature of transcription in eukaryotes is that the 
mechanisms that control its initiation are much more elabórate than 
those in prokaryotes—a point we discuss in detail in Chapter 8. In bac¬ 
teria, genes tend to lie very cióse to one another in the DNA, with only 
very short lengths of nontranscribed DNA between them. But in plants 
and animáis, including humans, individual genes are spread out along 
the DNA, with stretches of up to 100,000 nucleotide pairs between one 
gene and the next. This architecture allows a single gene to be con- 
trolled by a large variety of regulatory DNA sequerrces scattered along 
the DNA, and it enables eukaryotes to engage in more complex forms 
of transcriptional regulation than do bacteria. 

• Last but not least, eukaryotic transcription initiation must take into 
account the packing of DNA into nucleosomes and more compact 
forms of chromatin structure, as we describe in Chapter 8. 

We now turn to the general transcription factors and discuss how they 
help eukaryotic RNA polymerase II initiate transcription. 

Eukaryotic RNA Polymerase Requires General 
Transcription Factors 

The initial finding that, unlike bacterial RNA polymerase, purified eukaiy- 
otic RNA polymerase II could not initiate transcription on its own in a 
test tube led to the discovery and purification of the general transcrip¬ 
tion factors. These accessoiy proteins assemble on the promoter, where 
they position the RNA polymerase and pulí apart the DNA double helix 
to expose the témplate strand, allowing the polymerase to begin tran¬ 
scription. Thus the general transcription factors ha ve a similar role in 
eukaryotic transcription as sigma factor has in bacterial transcription. 
Figure 7-12 shows how the general transcription factors assemble at 
a promoter used by RNA polymerase II. The assembly process typically 
begins with the binding of the general transcription factor TFIID to a short 

Figure 7-12 To begin transcription, eukaryotic RNA polymerase II 
requires a set of general transcription factors. These transcription 
factors are called TFIIB, TFIID, and so on. (A) Many eukaryotic 
promoters contain a DNA sequence called the TATA box. (B) The 
TATA box is recognized by a subunit of the general transcription factor 
TFIID, called the TATA-binding protein (TBP). For simplicity, the DNA 
distortion produced by the binding of the TBP (see Figure 7-13) is 
not shown. (C) The binding of TFIID enables the adjacent binding of 
TFIIB. (D) The rest of the general transcription factors, as well as the 
RNA polymerase itself, assemble at the promoter. (E) TFIIH then pries 
apart the double helix at the transcription start point, using the energy 
of ATP hydrolysis, which exposes the témplate strand of the gene (not 
shown). TFIIH also phosphorylates RNA polymerase II, releasing the 
polymerase from most of the general transcription factors, so it can 
begin transcription. The site of phosphorylation is a long polypeptide 
"tail" that extends from the polymerase. 


Could the RNA polymerase used 
for transcription be used as the 
polymerase that makes the RNA 
primer required for DNA replication 
(discussed in Chapter 6)? 

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CHAPTER 7 From DNAto Protein: How Cells Read the Genome 


Figure 7-14 Before they can be 
translated, mRNA molecules made in 
the nudeus must be exported to the 
cytosol via pores in the nuclear envelope 
(red arrows). Shown here ¡s a section of a 
liver cell nucleus. The nucleolus ¡s where 
ribosomal RNAs are synthesized and 
combined with proteins to form ribosomes, 
which are then exported to the cytoplasm. 
(From D.W. Fawcett, ATextbook of 
Histology, llth ed. Philadelphia: Saunders, 
1986. With permission from Elsevier.) 

Figure 7-13 TATA-binding protein (TBP) binds to the TATA box 
(indicated by letters) and bends the DNA double helix. The unique 
distortion of DNA caused by TBP, which ¡s a subunit of TFIID (see 
Figure 7-12), helps attract the other general transcription factors. 

TBP ¡s a single polypeptide chain that ¡s folded into two very similar 
domains (blue and green). The protein sits atop the DNA double helix 
like a saddle on a bucking horse (Movie 7.4). (Adapted from J.L. Kim 
et al., Nature 365:520-527, 1993. With permission from Macmillan 
Publishers Ltd.) 

segment of DNA double helix composed primarily of T and A nucleotides; 
because of its composition, this parí of the promoter is known as the TATA 
box. Upon binding to DNA, TFIID causes a dramatic local distortion in the 
DNA double helix (Figure 7-13), which helps to serve as a landmark for 
the subsequent assembly of other proteins at the promoter. The TATA box 
is a key component of many promoters used by RNA polymerase II, and 
it is typically located 25 nucleotides upstream from the transcription start 
site. Once TFIID has bound to the TATA box, the other factors assemble, 
along with RNA polymerase II, to form a complete transcription initiation 
complex. Although Figure 7-12 shows the general transcription factors 
piling onto the promoter in a certain order, the actual order of assembly 
probably differs from one promoter to the next. 

After RNA polymerase II has been positioned on the promoter, it must be 
released from the complex of general transcription factors to begin its task 
of making an RNA molecule. A key step in liberating the RNA polymer¬ 
ase is the addition of phosphate groups to its "tail" (see Figure 7-12E). 
This liberation is initiated by the general transcription factor TFIIH, which 
contains a protein kinase as one of its subunits. Once transcription has 
begun, most of the general transcription factors dissociate from the DNA 
and then are available to initiate another round of transcription with a 
new RNA polymerase molecule. When RNA polymerase II finishes tran- 
scribing a gene, it too is released from the DNA; the phosphates on its 
tail are stripped off by protein phosphatases, and the polymerase is then 
ready to find a new promoter. Only the dephosphorylated form of RNA 
polymerase II can initiate RNA synthesis. 

Eukaryotic mRNAs Are Processed ¡n the Nucleus 

Although the templating principie by which DNA is transcribed into RNA 
is the same in all organisms, the way in which the RNA transcripts are 
handled before they can be used by the cell to make protein differs greatly 
between bacteria and eukaryotes. Bacterial DNA lies directly exposed 
to the cytoplasm, which contains the ribosomes on which protein syn¬ 
thesis takes place. As an mRNA molecule in a bacterium starts to be 
synthesized, ribosomes immediately attach to the free 5' end of the RNA 
transcript and begin translating it into protein. 

In eukaryotic cells, by contrast, DNA is enclosed within the nucleus. 
Transcription takes place in the nucleus, but protein synthesis takes 
place on ribosomes in the cytoplasm. So, before a eukaryotic mRNA 
can be translated into protein, it must be transported out of the nucleus 
through small pores in the nuclear envelope (Figure 7-14). Before it can 
be exported to the cytosol, however, a eukaryotic RNA must go through 
several RNA processing steps, which inelude capping, splicing, and poly- 
adenylation, as we discuss shortly. These steps take place as the RNA is 
being synthesized. The enzymes responsible for RNA processing ride on 
the phosphorylated tail of eukaryotic RNA polymerase II as it synthesizes 
an RNA molecule (see Figure 7-12), and they process the transcript as it 
emerges from the polymerase (Figure 7-15). 

LibertadDigital \ 2015 

From DNA to RNA 


Different types of RNA are processed in different ways before leaving the 
nucleus. Two processing steps, capping and polyadenylation, occur only 
on RNA transcripts destined to become mRNA molecules (called precur¬ 
sor mRNAs, or pre-mRNAs). 

1. RNA capping modifies the 5' end of the RNA transcript, the end that 
is synthesized first. The RNA is capped by the addition of an atypical 
nucleotide—a guanine (G) nucleotide bearing a methyl group, which 
is attached to the 5' end of the RNA in an unusual way (Figure 7-16). 
This capping occurs after RNA polymerase II has produced about 25 
nucleotides of RNA, long before it has completed transcribing the 
whole gene. 

2. Polyadenylation provides a newly transcribed mRNA with a spe- 
cial structure at its 3' end. In contrast with bacteria, where the 3' end 
of an mRNA is simply the end of the chain synthesized by the RNA 
polymerase, the 3' end of a forming eukaryotic mRNA is first trimmed 
by an enzyme that cuts the RNA chain at a particular sequence of 
nucleotides. The transcript is then finished off by a second enzyme 
that adds a series of repeated adenine (A) nucleotides to the cut end. 
This poly-A tail is generally a few hundred nucleotides long (see Figure 

These two modifications—capping and polyadenylation—increase the 
stability of a eukaryotic mRNA molecule, facilítate its export from the 
nucleus to the cytoplasm, and generally mark the RNA molecule as an 
mRNA. They are also used by the protein-synthesis machinery to make 
sure that both ends of the mRNA are present and that the message is 
therefore complete before protein synthesis begins. 

In Eukaryotes, Protein-Coding Genes Are Interrupted by 
Noncoding Sequences Called Introns 

Most eukaryotic pre-mRNAs have to undergo an additional processing 
step before they are functional mRNAs. This step involves a far more 
radical modification of the pre-mRNA transcript than capping or poly¬ 
adenylation, and it is the consequence of a surprising feature of most 
eukaryotic genes. In bacteria, most proteins are encoded by an uninter- 
rupted stretch of DNA sequence that is transcribed into an mRNA that, 
without any further processing, can be translated into protein. Most pro- 
tein-coding eukaryotic genes, in contrast, have their coding sequences 
interrupted by long, noncoding, intervening sequences called introns. 
The scattered pieces of coding sequence—called expressed sequences or 

Figure 7-16 Eukaryotic pre-mRNA molecules are modified by 
capping and polyadenylation. (A) A eukaryotic mRNA has a cap 
at the 5' end and a poly-A tail at the 3' end. Note that not all of the 
RNA transcript shown codes for protein. (B) The structure of the 
5' cap. Many eukaryotic mRNA caps carry an additional modification: 
the 2'-hydroxyl group on the second ribose sugar in the mRNA is 
methylated (not shown). 

RNA capping and polyadenylation 

(I ■ 






| CH 3 



|AAAAA 150 _ 25 o 3, 
poly-A tail 

RNA polymerase II 




¡plicing factors 


Figure 7-15 Phosphorylation of the tail of 
RNA polymerase II allows RNA-processing 
proteins to assemble there. Note that the 
phosphates shown here are in addition to 
the ones required fortranscription initiation 
(see Figure 7-12). Capping, polyadenylation, 
and splicing are all modifications that occur 
during RNA processing in the nucleus. 

LibertadDigital \ 2015 



From DNAto Protein: How Cells Read the Genome 

coding región 

bacterial gene 

coding regions noncoding regions 

(exons) (introns) 

eukaryotic gene 

Figure 7-17 Eukaryotic and bacterial genes are organized differently. A bacterial 
gene consists of a single stretch of uninterrupted nucleotide sequence that encodes 
the amino acid sequence of a protein (or more than one protein). In contrast, the 
protein-coding sequences of most eukaryotic genes (exons) are ¡nterrupted by 
noncoding sequences (introns). Promoters for transcription are indicated in green. 

exons—are usually shorter than the introns, and they often represent 
only a small fraction of the total length of the gene (Figure 7-17). Introns 
range in length from a single nucleotide to more than 10,000 nucleotides. 
Some protein-coding eukaryotic genes lack introns altogether, and some 
have only a few; but most have many (Figure 7-18). Note that the terms 
"exon" and "intron" apply to both the DNA and the corresponding RNA 

Figure 7-18 Most protein- 
coding human genes are 
broken into múltiple exons 
and introns. (A) The P-globin 
gene, which encodes one of 
the subunits of the oxygen- 
carrying protein hemoglobin, 
contains 3 exons. (B) The 
Factor VIII gene, which 
encodes a protein (Factor VIH) 
that functions in the blood- 
clotting pathway, contains 26 
exons. Mutations in this large 
gene are responsible for the 
most prevalent form of the 
blood disorder hemophilia. 

Introns Are Removed From Pre-mRNAs by RNA Splicing 

To produce an mRNA in a eukaryotic cell, the entire length of the gene, 
introns as well as exons, is transcribed into RNA. After capping, and as 
RNA polymerase II continúes to transcribe the gene, the process of RNA 
splicing begins, in which the introns are removed from the newly synthe- 
sized RNA and the exons are stitched together. Each transcript ultimately 
receives a poly-A tail; in some cases, this happens after splicing, whereas 
in other cases, it occurs before the final splicing reactions have been 
completed. Once a transcript has been spliced and its 5' and 3' ends have 
been modified, the RNA is now a functional mRNA molecule that can 
leave the nucleus and be translated into protein. 

How does the cell determine which parts of the RNA transcript to remove 
during splicing? Unlike the coding sequence of an exon, most of the 
nucleotide sequence of an intron is unimportant. Although there is lit- 
tle overall resemblance between the nucleotide sequences of different 
introns, each intron contains a few short nucleotide sequences that act 
as cues for its removal from the pre-mRNA. These special sequences are 
found at or near each end of the intron and are the same or very similar in 
all introns (Figure 7-19). Guided by these sequences, an elabórate splic¬ 
ing machine cuts out the intron in the form of a "lariat" structure (Figure 
7-20) , formed by the reaction of the "A" nucleotide highlighted in red in 
Figures 7-19 and 7-20. 

human P-globin gene 


(A) nucleotide pairs 

human Factor VIII gene introns 

1 _ 5 10 / 14 \ 


200,000 nucleotide pairs 

LibertadDigital \ 2015 

From DNA to RNA 


sequences required for intron removal 

portion of 

Figure 7-19 Special nucleotide sequences in a pre-mRNA transcript signal the 
beginning and the end of an intron. Only the nucleotide sequences shown are 
required to remove an intron; the other positions in an intron can be occupied by 
any nucleotide. The special sequences are recognized primarily by small nuclear 
ribonucleoproteins (snRNPs), which directthe cleavage ofthe RNA atthe intron- 
exon borders and catalyze the covalent linkage ofthe exon sequences. Here, in 
addition to the standard symbols for nucleoides (A, C, G, U), R stands for either A 
or G; Y stands for either C or U; N stands for any nucleotide. The A shown in red 
forms the branch point ofthe lariat produced in the splicing reaction shown in Figure 
7-20. The distances along the RNA between the three splicing sequences are highly 
variable; however, the distance between the branch point and the 5' splice junction is 
typically much longer than that between the 3' splice junction and the branch point 
(see Figure 7-20). The splicing sequences shown are from humans; similar sequences 
direct RNA splicing in other eukaryotes. 

We will not describe the splicing machineiy in detail, but it is worthwhile 
to note that, unlike the other steps of mRNA production we have dis- 
cussed, RNA splicing is carried out largely by RNA molecules rather than 
proteins. These RNA molecules, called small nuclear RNAs (snRNAs), 
are packaged with additional proteins to form small nuclear ribonucleo¬ 
proteins (snRNPs, pronounced "snurps"). The snRNPs recognize splice-site 
sequences through complementary base-pairing between their RNA 
components and the sequences in the pre-mRNA, and they also particí¬ 
pate intimately in the chemistry of splicing (Figure 7-21). Together, these 
snRNPs form the core of the spliceosome, the large assembly of RNA and 
protein molecules that carries out RNA splicing in the nucleus. To watch 
the spliceosome in action, see Movie 7.5. 

The intron-exon type of gene arrangement in eukaryotes may, at first, 
seem wasteful. It does, however, have a number of important benefits. 
First, the transcripts of many eukaiyotic genes can be spliced in differ- 
ent ways, each of which can produce a distinct protein. Such altemative 
splicing thereby allows many different proteins to be produced from 
the same gene (Figure 7-22). About 9596 of human genes are thought to 
undergo alternative splicing. Thus RNA splicing enables eukaryotes to 
increase the already enormous coding potential of their genomes. 

RNA splicing also provides another advantage to eukaryotes, one that is 
likely to have been profoundly important in the early evolutionary history 
of genes. As we discuss in detail in Chapter 9, the intron-exon structure 
of genes is thought to have sped up the emergence of new and useful 
proteins: novel proteins appear to have arisen by the mixing and match- 
ing of different exons of preexisting genes, much like the assembly of a 
new type of machine from a kit of preexisting functional components. 
Indeed, many proteins in present-day cells resemble patchworks com- 
posed from a common set of protein pieces, called protein domains (see 
Figure 4-51). 

, portion of spliced 
5 3 pre . m RNA 

Figure 7-20 An intron in a pre-mRNA 
molecule forms a branched structure 
during RNA splicing. In the first step, the 
branch point adenine (red A) ¡n the intron 
sequence attacks the 5' splice site and cuts 
the sugar-phosphate backbone ofthe RNA 
at this point (this is the same A highlighted 
in red in Figure 7-19). In this process, the 
cut 5' end ofthe intron becomes covalently 
linked to the 2'-OH group ofthe ribose 
of the A nucleotide to form a branched 
structure. The free 3'-OH end ofthe exon 
sequence then reacts with the start of 
the next exon sequence, joining the two 
exons together into a continuous coding 
sequence and releasing the intron in the 
form of a lariat structure, which is eventually 
degraded in the nucleus. 

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CHAPTER 7 From DNAto Protein: How Cells Read the Genome 

Figure 7-21 Splicing is carried out by 
a collection of RNA-protein complexes 
called snRNPs. There are five snRNPs, 
called U1, U2, U4, U5, and U6. As shown 
here, U1 and U2 bind to the 5' splice site 
(U1) and the lariat branch point (U2) through 
complementary base-pairing. Additional 
snRNPs are attracted to the splice site, 
and ¡nteractions between their protein 
components drive the assembly of the 
complete spliceosome. Rearrangements 
in the base pairs that hold together the 
snRNPs and the RNA transcript then 
reorganize the spliceosome to form the 
active site that excises the intron, 
leaving the spliced mRNA behind 
(see also Figure 7-20). 

RNA portion of snRNP base-pairs 

portion of 


portion of spliced mRNA 

Mature Eukaryotic mRNAs Are Exported from the Nucleus 

We have seen how eukaryotic pre-mRNA synthesis and processing take 
place in an orderly fashion within the cell nucleus. However, these events 
create a special problem for eukaryotic cells: of the total number of pre- 
mRNA transcripts that are synthesized, only a small fraction—the mature 
mRNAs—will be useful to the cell. The remaining RNA fragments— 
excised introns, broken RNAs, and aberrantly spliced transcripts—are not 
only useless, but they could be dangerous to the cell if allowed to leave 
the nucleus. How, then, does the cell distinguish between the relatively 
rare mature mRNA molecules it needs to export to the cytosol and the 
overwhelming amount of debris generated by RNA processing? 

The answer is that the transport of mRNA from the nucleus to the 
cytosol, where mRNAs are translated into protein, is highly selective: 
only correctly processed mRNAs are exported. This selective transport 
is mediated by nuclear pore complexes, which connect the nucleoplasm 
with the cytosol and act as gates that control which macromolecules can 
enter or leave the nucleus (discussed in Chapter 15). To be "export ready," 
an mRNA molecule must be bound to an appropriate set of proteins, each 
of which recognizes different parís of a mature mRNA molecule. These 
proteins inelude poly-A-binding proteins, a cap-binding complex, and 

Figure 7-22 Some pre-mRNAs undergo 
alternative RNA splicing to produce 
various mRNAs and proteins from the 
same gene. Whereas all exons are present 
in a pre-mRNA, some exons can be 
excluded from the final mRNA molecule. In 
this example, three of four possible mRNAs 
are produced. The 5' caps and poly-A tails 
on the mRNAs are not shown. 


three alternative mRNAs 

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From DNA to RNA 




proteins that bind to mRNAs that have been appropriately spliced (Figure 
7-23) . The entire set of bound proteins, rather than any single protein, 
ultimately determines whether an mRNA molecule will leave the nucleus. 
The "waste RNAs" that remain behind in the nucleus are degraded there, 
and their nucleotide building blocks are reused for transcription. 

mRNA Molecules Are Eventually Degraded in the Cytosol 

Because a single mRNA molecule can be translated into protein many 
times (see Figure 7-2), the length of time that a mature mRNA mole¬ 
cule persists in the cell affects the amount of protein it produces. Each 
mRNA molecule is eventually degraded into nucleotides by ribonucleases 
(RNAses) present in the cytosol, but the lifetimes of mRNA molecules dif- 
fer considerably—depending on the nucleotide sequence of the mRNA 
and the type of cell. In bacteria, most mRNAs are degraded rapidly, hav- 
ing a typical lifetime of about 3 minutes. The mRNAs in eukaiyotic cells 
usually persist longer: some, such as those encoding p-globin, have life¬ 
times of more than 10 hours, whereas others have lifetimes of less than 
30 minutes. 

These different lifetimes are in part controlled by nucleotide sequences 
in the mRNA itself, most often in the portion of RNA called the 3' untrans- 
lated región, which lies between the 3' end of the coding sequence and 
the poly-A tail. The different lifetimes of mRNAs help the cell control the 
amount of each protein that it synthesizes. In general, proteins made in 
large amounts, such as p-globin, are translated from mRNAs that have 
long lifetimes, whereas proteins made in smaller amounts, or whose lev¬ 
éis must change rapidly in response to signáis, are typically synthesized 
from short-lived mRNAs. 

The Earliest Cells May Have Had Introns ¡n Their Genes 

The process of transcription is universal: all cells use RNA polymerase 
and complementaiy base-pairing to synthesize RNA from DNA. Indeed, 
bacterial and eukaryotic RNA polymerases are almost identical in overall 
structure and clearly evolved from a shared ancestral polymerase. It may 
therefore seem puzzling that the resulting RNA transcripts are handled so 
differently in eukaiyotes and in prokaryotes (Figure 7-24). In particular, 
RNA splicing seems to mark a fundamental difference between those two 
types of cells. But how did this dramatic difference arise? 

As we have seen, RNA splicing provides eukaiyotes with the ability to 
produce a variety of proteins from a single gene. It also allows them 
to evolve new genes by mixing-and-matching exons from preexisting 
genes, as we discuss in Chapter 9. However, these advantages come with 
a cost: the cell has to maintain a larger genome and has to discard a 

Figure 7-23 A specialized set of RNA- 
binding proteins signáis that a mature 
mRNA is ready for export to the cytosol. 

As ¡ndicated on the left, the cap and 
poly-A tail of a mature mRNA molecule 
are "marked" by proteins that recognize 
these modifications. In addition, a group of 
proteins called the exon junction complex 
is deposited on the pre-mRNA after each 
successful splice has occurred. Once the 
mRNA is deemed "export ready," a nuclear 
transport receptor (discussed in Chapter 
15) associates with the mRNA and guides it 
through the nuclear pore. In the cytosol, the 
mRNA can shed some of these proteins and 
bind new ones, which, along with poly-A- 
binding protein, act as initiation factors for 
protein synthesis, as we discuss later. 

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CHAPTER 7 From DNAto Protein: How Cells Read the Genome 





Figure 7-24 Prokaryotes and eukaryotes 
handle their RNA transcripts differently. 

(A) In eukaryotic cells, the pre-mRNA 
molecule produced by transcriptlon 
contalns both ¡ntron and exon sequences. 

Its two ends are modlfied, and the introns 
are removed by RNA spliclng. The resulting 
mRNA is then transponed from the nucleus 
to the cytoplasm, where it is translated ¡nto 
protein. Although these steps are deplcted 
as occurrlng in sequence, one at a time, 

¡n reallty they occur simultaneously. For 
example, the RNA cap is usually added and 
splicing usually begins before transcription 
has been completed. Because of this 
overlap, transcripts of the entlre gene 
(including all Introns and exons) do not 
typlcally exlst in the cell. (B) In prokaryotes, 
the productlon of mRNA molecules 
Is slmpler. The 5' end of an mRNA 
molecule ¡s produced by the ¡nltiatlon of 
transcriptlon by RNA polymerase, and the 
3' end ¡s produced by the terminatlon of 
transcriptlon. Because prokaryotlc cells 
lack a nucleus, transcriptlon and translatlon 
take place in a common compartment. 
Translatlon of a bacterial mRNA can 
therefore begin before ¡ts synthesls has 
been completed. In both eukaryotes and 
prokaryotes, the amount of a protein ¡n a 
cell depends on the rates of each of these 
steps, as well as on the rates of degradaron 
of the mRNA and protein molecules. 

large fraction of the RNA it synthesizes without ever using it. According 
to one school of thought, early cells—the common ancestors of prokary¬ 
otes and eukaryotes—contained introns that were lost in prokaryotes 
during subsequent evolution. By shedding their introns and adopting a 
smaller, more streamlined genome, prokaryotes would have been able to 
reproduce more rapidly and efflciently. Consistent with this idea, simple 
eukaryotes that reproduce rapidly (some yeasts, for example) have rela- 
tively few introns, and these introns are usually much shorter than those 
found in higher eukaryotes. 

On the other hand, some argüe that introns were originally parasitic 
mobile genetic elements (discussed in Chapter 9) that happened to 
invade an early eukaryotic ancestor, colonizing its genome. These host 
cells then unwittingly replicated the "stowaway" nucleotide sequences 
along with their own DNA; modern eukaryotes simply never bothered 
to sweep away the genetic clutter left from that ancient infection. The 
issue, however, is far from settled; whether introns evolved early—and 
were lost in prokaryotes—or evolved later in eukaryotes is still a topic of 
scientific debate, and we return to it in Chapter 9. 


By the end of the 1950s, biologists had demonstrated that the informa- 
tion encoded in DNA is copied first into RNA and then into protein. The 
debate then shifted to the "coding problem": How is the information in 
a linear sequence of nucleotides in an RNA molecule translated into the 
linear sequence of a chemically quite different set of subunits—the amino 
acids in a protein? This fascinating question intrigued scientists at the 
time. Here was a cryptogram set up by nature that, after more than 3 
billion years of evolution, could finally be solved by one of the producís 
of evolution—human beings! Indeed, scientists have not only cracked 
the code but have revealed, in atomic detail, the precise workings of the 
machinery by which cells read this code. 

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From RNA to Protein 












Ala Arg Asp Asn Cys Glu Gln Gly His lie Leu Lys Met Phe Pro Ser Thr Trp Tyr Val stop 

Figure 7-25 The nucleotide sequence of an mRNA is translated into the amino acid sequence of a protein via the genetic code. 

All the three-nucleotide codons ¡n mRNAs that specify a given amino acid are listed above that amino acid, which is given in both ¡ts 
three-letter and one-letter abbreviations (see Panel 2-5, pp. 74-75, for the full ñame of each amino acid and ¡ts structure). Like RNA 
molecules, codons are always written with the 5'-terminal nucleotide to the left. Note that most amino acids are represented by more 
than one codon, and there are some regularities in the set of codons that specify each amino acid. Codons for the same amino acid tend 
to contain the same nucleotides at the first and second positions and to vary at the third position. There are three codons that do not 
specify any amino acid but act as termination sites (stop codons ), signaling the end of the protein-coding sequence in an mRNA. One 
codon—AUG—acts both as an initiation codon, signaling the start of a protein-coding message, and as the codon that specifies the 
amino acid methionine. 

An mRNA Sequence Is Decoded ¡n Sets of Three 

Transcription as a means of information transfer is simple to understand: 
DNA and RNA are chemically and structurally similar, and DNA can act as 
a direct témplate for the synthesis of RNA through complementary base- 
pairing. As the term transcription signifies, it is as if a message written 
out by hand were being converted, say, into a typewritten text. The lan- 
guage itself and the form of the message do not change, and the symbols 
used are closely related. 

In contrast, the conversión of the information in RNA into protein rep- 
resents a translation of the information into another language that 
uses different symbols. Because there are only 4 different nucleotides in 
mRNA but 20 different types of amino acids in a protein, this translation 
cannot be accounted for by a direct one-to-one correspondence between 
a nucleotide in RNA and an amino acid in protein. The rules by which the 
nucleotide sequence of a gene, through an intermediary mRNA molecule, 
is translated into the amino acid sequence of a protein are known as the 
genetic code. 

In 1961, it was discovered that the sequence of nucleotides in an mRNA 
molecule is read consecutively in groups of three. And because RNA is 
made of 4 different nucleotides, there are 4 x 4 x 4 = 64 possible combi- 
nations of three nucleotides: AAA, AUA, AUG, and so on. However, only 
20 different amino acids are commonly found in proteins. Either some 
nucleotide triplets are never used, or the code is redundant, with some 
amino acids being specified by more than one triplet. The second pos- 
sibility turned out to be correct, as shown by the completely deciphered 
genetic code shown in Figure 7-25. Each group of three consecutive 
nucleotides in RNA is called a codon, and each codon specifies one 
amino acid. The strategy by which this code was cracked is described in 
How We Know, pp. 240-241. 

The same genetic code is used in nearly all present-day organisms. 
Although a few slight differences have been found, these occur chiefly in 
the mRNA of mitochondria and of some fungi and protozoa. Mitochondria 
have their own DNA replication, transcription, and protein-synthe- 
sis machinery, which operates independently from the corresponding 
machineiy in the rest of the cell (discussed in Chapter 14), and they have 
been able to accommodate minor changes to the otherwise universal 
genetic code. Even in fungi and protozoa, the similarities in the code far 
outweigh the differences. 

In principie, an mRNA sequence can be translated in any one of three dif¬ 
ferent reading frames, depending on where the decoding process begins 
(Figure 7-26). However, only one of the three possible reading frames 

i cuc i, AGC i 

— Leu-Ser-Val-Thr — 

— Ser-Ala-Leu-Pro — 

i CAG i i CG U i 

— Gln-Arg-Tyr-His — 

Figure 7-26 In principie, an mRNA 
molecule can be translated in three 
possible reading frames. In the process of 
translating a nucleotide sequence (b/ue) into 
an amino acid sequence (red), the sequence 
of nucleotides in an mRNA molecule is read 
from the 5' to the 3' end in sequential sets 
of three nucleotides. In principie, therefore, 
the same mRNA sequence can specify three 
completely different amino acid sequences, 
depending on where translation begins— 
that is, on the reading frame used. In reality, 
however, only one of these reading frames 
encodes the actual message and is therefore 
used in translation, as we discuss later. 

LibertadDigital \ 2015 




By the beginning of the 1960s, the central dogma had 
been accepted as the pathway along which informa- 
tion flows from gene to protein. It was clear that genes 
encode proteins, that genes are made of DNA, and that 
mRNA serves as an intermediary, carrying the infor- 
mation from DNA to the ribosome, where the RNA is 
translated into protein. 

Even the general format of the genetic code had been 
worked out: each of the 20 amino acids found in pro¬ 
teins is represented by a triplet codon in an mRNA 
molecule. But an even greater challenge remained: 
biologists, chemists, and even physicists set their sights 
on breaking the genetic code—attempting to figure out 
which amino acid each of the 64 possible nucleotide tri- 
plets designates. The most straightforward path to the 
solution would have been to compare the sequence of 
a segment of DNA or of mRNA with its corresponding 
polypeptide product. Techniques for sequencing nucleic 
acids, however, would not be devised for another 10 

So researchers decided that, to crack the genetic code, 
they would have to synthesize their own simple RNA 
molecules. If they could feed these RNA molecules to 
ribosomes—the machines that make proteins—and 
then analyze the resulting polypeptide product, they 
would be on their way to deciphering which triplets 
encode which amino acids. 

Losing the cells 

Before researchers could test their synthetic mRNAs, 
they needed to perfect a cell-free system for protein 
synthesis. This would allow them to transíate their 
messages into polypeptides in a test tube. (Generally 
speaking, when working in the laboratory, the simpler 
the system, the easier it is to interpret the results.) To 
isolate the molecular machineiy they needed for such 
a cell-free translation system, researchers broke open 
E. coli cells and loaded their contents into a centrifuge 
tube. Spinning these samples at high speed caused the 
membranes and other large chunks of cellular debris to 
be dragged to the bottom of the tube; the lighter cellular 
components required for protein synthesis—including 
mRNA, the tRNA adaptors, ribosomes, enzymes, and 
other small molecules—were left floating in the supema- 
tant. Researchers found that simply adding radioactive 
amino acids to this cell "soup" would trigger the produc- 
tion of radiolabeled polypeptides. By centrifuging this 
supematant again, at a higher speed, the researchers 
could forcé the ribosomes, and any newly synthesized 
peptides attached to them, to the bottom of the tube; the 
labeled polypeptides could then be detected by measur- 
ing the radioactivity in the sediment remaining in the 
tube after the top layer had been discarded. 

The trouble with this particular system was that it 
produced proteins encoded by the cell's own mRNAs 
already present in the extract. But researchers wanted 
to use their own synthetic messages to direct protein 
synthesis. This problem was solved when Marshall 
Nirenberg discovered that he could destroy the cells' 
mRNA in the extract by adding a small amount of ribo- 
nuclease—an enzyme that degrades RNA—to the mix. 
Now all he needed to do was prepare large quantities of 
synthetic mRNA, add it to the cell-free system, and see 
what peptides carne out. 

Faking the message 

Producing a synthetic polynucleotide with a defined 
sequence was not as simple as it sounds. Again, it 
would be years before chemists and bioengineers devel- 
oped machines that could synthesize any given string 
of nucleic acids quickly and cheaply. Nirenberg decided 
to use polynucleotide phosphorylase, an enzyme that 
would join ribonucleotides together in the absence of a 
témplate. The sequence of the resulting RNA would then 
depend entirely on which nucleotides were presented 
to the enzyme. A mixture of nucleotides would be sewn 
into a random sequence; but a single type of nucleotide 
would yield a homogeneous polymer containing only 
that one nucleotide. Thus Nirenberg, working with his 
collaborator Heinrich Matthaei, first produced synthetic 
mRNAs made entirely of uracil—poly U. 

Together, the researchers fed this poly U to their cell- 
free translation system. They then added a single type 
of radioactively labeled amino acid to the mix. After 
testing each amino acid—one at a time, in 20 differ- 
ent experiments—they determined that poly U directs 
the synthesis of a polypeptide containing only phenyl- 
alanine (Figure 7-27). With this electrifying result, the 
first word in the genetic code had been deciphered (see 
Figure 7-25). 

Nirenberg and Matthaei then repeated the experiment 
with poly A and poly C and determined that AAA codes 
for lysine and CCC for proline. The meaning of poly G 
could not be ascertained by this method because this 
polynucleotide forms an odd triple-stranded helix that 
did not serve as a témplate in the cell-free system. 
Feeding ribosomes with synthetic RNA seemed a 
fruitful technique. But with the single-nucleotide pos- 
sibilities exhausted, researchers had nailed down only 
three codons; they had 61 still to go. The other codons, 
however, were harder to decipher, and a new synthetic 
approach was needed. In the 1950s, the organic chem- 
ist Gobind Khorana had been developing methods for 
preparing mixed polynucleotides of defined sequence— 
but his techniques worked only for DNA. When he 

LibertadDigital \ 2015 

From RNA to Protein 


synthetic mRNA 

N Phe Phe Phe Phe Phe Phe Phe Phe C 
radioactive polypeptide synthesized 

cell-free translation 
System plus radioactive 
amino acids 

Figure 7-27 UUU codes for 
phenylalanine. Synthetic mRNAs 
are fed into a cell-free translation 
system containing bacterial 
rlbosomes, tRNAs, enzymes, 
and othersmall molecules. 
Radioactive amino acids are 
added to this mix and the 
resulting polypeptides analyzed. 
In this case, poly U ¡s shown to 
encode a polypeptide containing 
only phenylalanine. 

learned of Nirenberg's work with synthetic RNAs, 
Khorana directed his energies and skills to producing 
polyribonucleotides. He found that if he started out by 
making DNAs of a defined sequence, he could then use 
RNA polymerase to produce RNAs from those. In this 
way, Khorana prepared a collection of different RNAs 
of defined repeating sequence: he generated sequences 
of repeating dinucleotides (such as poly UC), trinucleo- 
tides (such as poly UUC), or tetranucleotides (such as 

These mixed polynucleotides, however, yielded results 
that were much more difficult to decode than the mono- 
nucleotide messages that Nirenberg had used. Take poly 
UG, for example. When this repeating dinucleotide is 
added to the translation system, researchers discovered 
that it codes for a polypeptide of alternating cysteines 
and valines. This RNA, of course, contains two differ¬ 
ent alternating codons: UGU and GUG. So researchers 
could say that UGU and GUG code for cysteine and 
valine, although they could not tell which went with 
which. Thus these mixed messages provided useful 
information, but they did not definitively reveal which 
codons specified which amino acids (Figure 7-28). 

trinucleotides bound to the ribosomes, and Phe-tRNAs 
bound to the UUU. The new system was up and running, 
and the researchers had confirmed that UUU codes for 

All that remained was for researchers to produce all 64 
possible codons—a task that was quickly accomplished 
in both Nirenberg's and Khorana’s laboratories. Because 
these small trinucleotides were much simpler to syn- 
thesize chemically, and the triplet-trapping tests were 
easier to perform and analyze than the previous decod- 
ing experiments, the researchers were able to work out 
the complete genetic code within the next year. 






poly UG ...Cys-Val-Cys-Val... 

poIyAG ...Arg-Glu-Arg-Glu... 

KÍ« W| - 

gag]~ Ar9, Glu 

poly UUC 





Trapping the triplets 

These final ambiguities in the code were resolved when 
Nirenberg and a young medical gradúate named Phil 
Leder discovered that RNA fragments that were only 
three nucleotides in length—the size of a single codon— 
could bind to a ribosome and attract the appropriate 
amino-acid-containing tRNA molecule to the protein- 
making machinery. These complexes—containing one 
ribosome, one mRNA codon, and one radiolabeled 
aminoacyl-tRNA—could then be captured on a piece of 
filter paper and the attached amino acid identified. 

Their trial run with UUU—the first word—worked 
splendidly. Leder and Nirenberg primed the usual cell- 
free translation system with snippets of UUU. These 


poly UAUC ...Tyr-Leu-Ser-lle... CUA 

* One codon specifies Cys, the other Val, but which is which? 

The same ambiguity exists for the other codon assignments 
shown here. 

Figure 7-28 Using synthetic RNAs of mixed, repeating 
ribonucleotide sequences, scientists further narrowed 
the coding possibilities. Although these mixed messages 
produced mixed polypeptides, they did not permit the 
unambiguous assignment of a single codon to a specific amino 
acid. For example, the results of the poly-UG experiment 
cannot distinguish whether UGU or GUG encodes cysteine. 

As indicated, the same type of ambiguity confounded the 
interpretation of all the experiments using di-, tri-, and 

UbertadDigital \ 2015 

242 CHAPTER 7 From DNA to Protein: How Cells Read the Genome 

in an mRNA specifies the correct protein. We discuss later how a special 
punctuation signal at the beginning of each mRNA molecule sets the cor¬ 
rect reading frame. 

tRNA Molecules Match Amino Acids to Codons in mRNA 

The codons in an mRNA molecule do not directly recognize the amino 
acids they specify: the group of three nucleotides does not, for exam- 
ple, bind directly to the amino acid. Rather, the translation of mRNA into 
protein depends on adaptor molecules that can recognize and bind to a 
codon at one site on their surface and to an amino acid at another site. 
These adaptors consist of a set of small RNA molecules known as trans- 
fer RNAs (tRNAs), each about 80 nucleotides in length. 

We saw earlier that an RNA molecule generally folds into a three-dimen- 
sional structure by forming base pairs between different regions of the 
molecule. If the base-paired regions are sufficiently extensive, they will 
fold back on themselves to form a double-helical structure, like that of 
double-stranded DNA. The tRNA molecule provides a striking example of 
this. Four short segments of the folded tRNA are double-helical, producing 
a molecule that looks like a cloverleaf when drawn schematically (Figure 
7-29A). For example, a 5'-GCUC-3' sequence in one part of a polynucle- 
otide chain can base-pair with a 5'-GAGC-3' sequence in another región 
of the same molecule. The cloverleaf undergoes further folding to form a 
compact, L-shaped structure that is held together by additional hydrogen 
bonds between different regions of the molecule (Figure 7-29B and C). 
Two regions of unpaired nucleotides situated at either end of the L-shaped 
tRNA molecule are crucial to the function of tRNAs in protein synthesis. 


(E) anticodon 

Figure 7-29 tRNA molecules are molecular adaptors, linking amino acids to codons. In this series of diagrams, the same tRNA 
molecule—in this case, a tRNA specific for the amino acid phenylalanine (Phe)—is depicted in various ways. (A) The conventional 
"cloverleaf" structure shows the complementary base-pairlng (red Unes) that creates the double-helical regions of the molecule. The 
anticodon loop (b/ue) contains the sequence of three nucleotides (red letters) that base-pairs with a codon in mRNA. The amino acid 
matching the codon-anticodon pair is attached at the 3' end of the tRNA. tRNAs contain some unusual bases, which are produced by 
Chemical modlfication afterthe tRNA has been syntheslzed. The bases denoted Y (for pseudourldine) and D (for dihydrouridine) are 
derived from uracll. (B and C) Views of the actual L-shaped molecule, based on X-ray diffraction analysis. These two images are rotated 
90° with respect to each other. (D) Schematic representation of tRNA, emphasizing the anticodon, that will be used in subsequent 
figures. (E) The linear nucleotide sequence of the tRNA molecule, color-coded to match A, B, and C. 

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From RNA to Protein 


in an mRNA molecule. The other is a short single-stranded región at the 
3' end of the molecule; this is the site where the amino acid that matches 
the codon is covalently attached to the tRNA. 

We saw in the previous section that the genetic code is redundant; that 
is, several different codons can specify a single amino acid (see Figure 
7-25). This redundancy implies either that there is more than one tRNA 
for many of the amino acids or that some tRNA molecules can base-pair 
with more than one codon. In fact, both situations occur. Some amino 
acids have more than one tRNA, and some tRNAs are constructed so 
that they require accurate base-pairing only at the first two positions of 
the codon and can tolérate a mismatch (or wobble) at the third position. 
This wobble base-pairing explains why so many of the alternative codons 
for an amino acid differ only in their third nucleotide (see Figure 7-25). 
Wobble base-pairings make it possible to flt the 20 amino acids to their 
61 codons with as few as 31 kinds of tRNA molecules. The exact number 
of different kinds of tRNAs, however, differs from one species to the next. 
For example, humans have nearly 500 different tRNA genes, but only 48 
anticodons are represented among them. 

Specific Enzymes Couple tRNAs to the Corred Amino 

For a tRNA molecule to cariy out its role as an adaptor, it must be linked— 
or charged—with the correct amino acid. How does each tRNA molecule 
recognize the one amino acid in 20 that is its right partner? Recognition 
and attachment of the correct amino acid depend on enzymes called 
aminoacyl-tRNA synthetases, which covalently couple each amino acid 
to its appropriate set of tRNA molecules. In most organisms, there is a 
different synthetase enzyme for each amino acid. That means that there 
are 20 synthetases in all: one attaches glycine to all tRNAs that recog¬ 
nize codons for glycine, another attaches phenylalanine to all tRNAs that 
recognize codons for phenylalanine, and so on. Each synthetase enzyme 
recognizes specific nucleotides in both the anticodon and the amino- 
acid-accepting arm of the correct tRNA (Movie 7.6). The synthetases are 
thus equal in importance to the tRNAs in the decoding process, because 
it is the combined action of the synthetases and tRNAs that allows each 
codon in the mRNA molecule to specify its proper amino acid (Figure 

Figure 7-30 The genetic code is 
translated by the cooperation of two 
adaptors: aminoacyl-tRNA synthetases 
and tRNAs. Each synthetase couples a 
particular amino acid to its corresponding 
tRNAs, a process called charging. The 
anticodon on the charged tRNA molecule 
then forms base pairs with the appropriate 
codon on the mRNA. An error in either 
the charging step orthe binding of the 
charged tRNA to its codon will cause the 
wrong amino acid to be incorporated into 
a protein chain. In the sequence of events 
shown, the amino acid tryptophan (Trp) is 
selected by the codon UGG on the mRNA. 


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From DNAto Protein: How Cells Read the Genome 


ln a clever experiment performed in 
1962, a cysteine already attached to 
its tRNA was chemically converted 
to an alanine. These "hybrid" tRNA 
molecules were then added to a cell- 
free translation system from which 
the normal cysteine-tRNAs had 
been removed. When the resulting 
protein was analyzed, it was found 
that alanine had been inserted at 
every point in the polypeptide chain 
where cysteine was supposed to be. 
Discuss what this experiment tells 
you about the role of aminoacyl- 
tRNA synthetases during the normal 
translation of the genetic code. 

The synthetase-catalyzed reaction that attaches the amino acid to the 3' 
end of the tRNA is one of many reactions in cells coupled to the energy- 
releasing hydrolysis of ATP (see Figure 3-33). The reaction produces a 
high-energy bond between the charged tRNA and the amino acid. The 
energy of this bond is later used to link the amino acid covalently to the 
growing polypeptide chain. 

The mRNA Message Is Decoded by Ribosomes 

The recognition of a codon by the anticodon on a tRNA molecule depends 
on the same type of complementaiy base-pairing used in DNA replication 
and transcription. However, accurate and rapid translation of mRNA into 
protein requires a molecular machine that can move along the mRNA, 
capture complementaiy tRNA molecules, hold the tRNAs in position, and 
then covalently link the amino acids that they carry to form a polypeptide 
chain. In both prokaiyotes and eukaryotes, the machine that gets the job 
done is the ribosome—a large complex made from dozens of small pro- 
teins (the ribosomal proteins) and several crucial RNA molecules called 
ribosomal RNAs (rRNAs). A typical eukaryotic cell contains millions of 
ribosomes in its cytoplasm (Figure 7-31). 

Eukaryotic and prokaryotic ribosomes are very similar in structure and 
function. Both are composed of one large subunit and one small subunit, 
which fit together to form a complete ribosome with a mass of several 
million daltons (Figure 7-32); for comparison, an average-sized pro¬ 
tein has a mass of 30,000 daltons. The small ribosomal subunit matches 
the tRNAs to the codons of the mRNA, while the large subunit catalyzes 
the formation of the peptide bonds that covalently link the amino acids 
together into a polypeptide chain. These two subunits come together on 
an mRNA molecule near its 5' end to start the synthesis of a protein. The 
mRNA is then pulled through the ribosome like a long piece of tape. As 
the mRNA inches forward in a 5'-to-3' direction, the ribosome translates 
its nucleotide sequence into an amino acid sequence, one codon at a 
time, using the tRNAs as adaptors. Each amino acid is thereby added in 
the correct sequence to the end of the growing polypeptide chain (Movie 
7.7). When synthesis of the protein is finished, the two subunits of the 
ribosome sepárate. Ribosomes opérate with remarkable efficiency: a 
eukaryotic ribosome adds about 2 amino acids to a polypeptide chain 
each second; a bacterial ribosome operates even faster, adding about 20 
amino acids per second. 

Figure 7-31 Ribosomes are located in 
the cytoplasm of eukaryotic cells. This 
electrón micrograph shows a thin section of 
a small región of cytoplasm. The ribosomes 
appear as small gray blobs. Some are free in 
the cytosol (red arrows); others are attached 
to membranes of the endoplasmic reticulum 
(green arrows). (Courtesy of George Palade.) 

LibertadDigital \ 2015 

From RNAto Protein 


MW = 2,800,000 




+ c 

••• • 

• • •• 

-33 ribosomal proteins + 

Figure 7-32 The eukaryotic ribosome 
is a large complex of four rRNAs and 
more than 80 small proteins. Prokaryotic 
ribosomes are very similar: both are formed 
from a large and small subunit, which only 
come together after the small subunit has 
bound an mRNA. Although ribosomal 
proteins greatly outnumber rRNAs, the 
RNAs account for most of the mass of the 
ribosome and give ¡t its overall shape and 

complete eukaryotic ribosome 
MW = 4,200,000 

How does the ribosome choreograph all the movements required for 
translation? In addition to a binding site for an mRNA molecule, each 
ribosome contains three binding sites for tRNA molecules, called the A 
site, the P site, and the E site (Figure 7-33). To add an amino acid to a 
growing peptide chain, the appropriate charged tRNA enters the A site 
by base-pairing with the complementary codon on the mRNA molecule. 
Its amino acid is then linked to the peptide chain held by the tRNA in the 
neighboring P site. Next, the large ribosomal subunit shifts forward, mov- 
ing the spent tRNA to the E site before ejecting it (Figure 7-34). This cycle 
of reactions is repeated each time an amino acid is added to the polypep- 
tide chain, with the new protein growing from its amino to its carboxyl 
end until a stop codon in the mRNA is encountered. 

Figure 7-33 Each ribosome has a binding 
site for mRNA and three binding sites 
for tRNA. The tRNA sites are designated 
the A, P, and E sites (short for aminoacyl- 
tRNA, peptidyl-tRNA, and exit, respectively). 
(A) Three-dimensional structure of a 
bacterial ribosome, as determined by X-ray 
crystallography, with the small subunit in 
dark green and the large subunit in Hght 
green. Both the rRNAs and the ribosomal 
proteins are shown in green. tRNAs are 
shown bound in the E site (red), the P site 
(orange), and the A site (yellow). Although 
all three tRNA sites are shown occupied 
here, during the process of protein synthesis 
only two of these sites are occupied at 
any one time (see Figure 7-34). (B) Highly 
schematized representation of a ribosome 
(in the same orientation as A), which will be 
used in subsequent figures. Note that both 
the large and small subunits are ¡nvolved 
in forming the A, P, and E sites, while only 
the small subunit forms the binding site for 
an mRNA. (B, adapted from M.M. Yusupov 
et al., Science 292:883-896, 2001, with 
permission from AAAS. Courtesy of Albion 
Baucom and Plarry Noller.) 

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CHAPTER 7 From DNAto Protein: How Cells Read the Genome 

Figure 7-35 Ribosomal RNAs give the 
ribosome its overall shape. Shown here 
are the detailed structures of the two rRNAs 
that form the core of the large subunit of 
a bacterial ribosome—the 23S rRNA ( blue ) 
and the 5S rRNA (purp/e). One of the protein 
subunits of the ribosome (L1) ¡s included 
as a reference point, as this protein forms 
a characteristic protrusion on the ribosome 
surface. Ribosomal components are commonly 
designated by their "S valúes," which 
referió their rate of sedimentation in an 
ultracentrifuge. (Adapted from N. Ban et al., 
Science 289:905-920, 2000. With permission 
from AAAS.) 

Figure 7-34 Translation takes place in a four-step cyde. This cycle 
¡s repeated over and over during the synthesis of a protein. In step 

1, a charged tRNA carrying the next amino acid to be added to the 
polypeptide chain binds to the vacant A site on the ribosome by forming 
base pairs with the mRNA codon that is exposed there. Because only 
the appropriate tRNA molecules can base-pair with each codon, this 
codon determines the specific amino acid added. The A and P sites are 
sufficiently cióse together that their two tRNA molecules are forced to 
form base pairs with codons that are contiguous, with no stray bases in 
between. This positioning of the tRNAs ensures that the correct reading 
frame will be preserved throughoutthe synthesis of the protein. In step 

2, the carboxyl end of the polypeptide chain (amino acid 3 in step 1) is 
uncoupled from the tRNA at the P site and joined by a peptide bond to 
the free amino group of the amino acid linked to the tRNA at the A site. 

This reaction is catalyzed by an enzymatic site in the large subunit. In 
step 3, a shift of the large subunit relative to the small subunit moves the 
two tRNAs into the E and P sites of the large subunit. In step 4, the small 
subunit moves exactly three nucleoides along the mRNA molecule, 
bringing it backto its original position relative to the large subunit. This 
movement ejects the spent tRNA and resets the ribosome with an empty 
A site so that the next charged tRNA molecule can bind (Movie 7.8). 

As indicated, the mRNA is translated in the 5'-to-3' direction, and the 
N-terminal end of a protein is made first, with each cycle adding one 
amino acid to the C-terminus of the polypeptide chain. To watch the 
translation cycle in atomic detail, see Movie 7.9. 

The Ribosome Is a Ribozyme 

The ribosome is one of the largest and most complex structures in the cell, 
composed of two-thirds RNA and one-third protein by weight. The deter- 
mination of the entire three-dimensional structure of its large and small 
subunits in 2000 was a major triumph of modern biology. The structure 
confirmed earlier evidence that the rRNAs—not the proteins—are respon- 
sible for the ribosome's overall structure and its ability to choreograph 
and catalyze protein synthesis. 

The rRNAs are folded into highly compact, precise three-dimensional 
structures that form the core of the ribosome (Figure 7-35). In marked 
contrast to the central positioning of the rRNAs, the ribosomal proteins 
are generally located on the surface, where they fill the gaps and crevices 
of the folded RNA. The main role of the ribosomal proteins seems to be 

LibertadDigital \ 2015 

From RNA to Protein 


to help fold and stabilize the RNA core, while permitting the changes in 
rRNA conformation that are necessary for this RNA to catalyze efficient 
protein synthesis. 

Not only are the three tRNA-binding sites (the A, P, and E sites) on the 
ribosome formed primarily by the rRNAs, but the catalytic site for peptide 
bond formation is formed by the 23S rRNA of the large subunit; the near- 
est ribosomal protein is located too far away to make contact with the 
incoming charged tRNA or with the growing polypeptide chain. The cata¬ 
lytic site in this rRNA—a peptidyl transferase—is similar in many respects 
to that found in some protein enzymes: it is a highly structured pocket 
that precisely orients the two reactants—the elongating polypeptide and 
the charged tRNA—thereby greatly increasing the probability of a produc- 
tive reaction. 

RNA molecules that possess catalytic activity are called ribozymes. Later, 
in the final section of this chapter, we will consider other ribozymes and 
discuss what the existence of RNA-based catalysis might mean for the 
early evolution of life on Earth. Here we need only note that there is good 
reason to suspect that RNA rather than protein molecules served as the 
first catalysts for living cells. If so, the ribosome, with its catalytic RNA 
core, could be viewed as a relie of an earlier time in life's history, when 
cells were run almost entirely by ribozymes. 

Specific Codons in mRNA Signal the Ribosome Where to 
Start and to Stop Protein Synthesis 

In the test tube, ribosomes can be forced to transíate any RNA molecule 
(see How We Know, pp. 240-241). In a cell, however, a specific start sig¬ 
nal is required to initiate translation. The site at which protein synthesis 
begins on an mRNA is crucial, because it sets the reading frame for the 
whole length of the message. An error of one nucleotide either way at 
this stage will cause every subsequent codon in the mRNA to be misread, 
resulting in a nonfunctional protein with a garbled sequence of amino 
acids (see Figure 7-26). And the rate of initiation determines the rate at 
which the protein is synthesized from the mRNA. 

The translation of an mRNA begins with the codon AUG, and a special 
charged tRNA is required to initiate translation. This initiator tRNA 
always carries the amino acid methionine (or a modified form of methio- 
nine, formyl-methionine, in bacteria). Thus newly made proteins all have 
methionine as the first amino acid at their N-terminal end, the end of a 
protein that is synthesized first. This methionine is usually removed later 
by a specific protease. 

In eukaryotes, an initiator tRNA, charged with methionine, is first loaded 
into the P site of the small ribosomal subunit, along with additional pro¬ 
teins called translation initiation factors (Figure 7-36). The initiator 
tRNA is distinct from the tRNA that normally carries methionine. Of all 
the tRNAs in the cell, only a charged initiator tRNA molecule is capable of 
binding tightly to the P site in the absence of the large ribosomal subunit. 
Next, the small ribosomal subunit loaded with the initiator tRNA binds to 

Figure 7-36 Initiation of protein synthesis ¡n eukaryotes requires 
translation initiation factors and a special initiator tRNA. Although 
not shown here, efficient translation initiation also requires additional 
proteins that are bound at the 5' cap and poly-A tail of the mRNA 
(see Figure 7-23). In this way, the translation apparatus can ascertain 
that both ends of the mRNA are intact before initiating translation. 
Following initiation, the protein is elongated by the reactions outlined 
in Figure 7-34. 


A sequence of nucleotides in a DNA 
was used as a témplate to 
synthesize an mRNA that was then 
translated into protein. Predict 
the C-terminal amino acid and 
the N-terminal amino acid of the 
resulting polypeptide. Assume that 
the mRNA is translated without the 
need for a start codon. 

translation initiation 

factors bound 


LibertadDigital \ 2015 


CHAPTER 7 From DNAto Protein: How Cells Read the Genome 

protein a protein (3 protein y 

Figure 7-37 A single prokaryotic mRNA molecule can encode several different 
proteins. In prokaryotes, genes directing the different steps ¡n a process are often 
organized ¡nto clusters (operons) that are transcribed together ¡nto a single mRNA. 

A prokaryotic mRNA does not have the same sort of 5' cap as a eukaryotic mRNA, 
but instead has a triphosphate at its 5' end. Prokaryotic ribosomes initiate translation 
at ribosome-binding sites (dark blue), which can be located in the interior of an 
mRNA molecule. This feature enables prokaryotes to synthesize different proteins 
from a single mRNA molecule, with each protein made by a different ribosome. 

the 5' end of an mRNA molecule, which is marked by the 5' cap that is 
present on all eukaryotic mRNAs (see Figure 7-16). The small ribosomal 
subunit then moves forward (5' to 3') along the mRNA searching for the 
first AUG. When this AUG is encountered and recognized by the initiator 
tRNA, several initiation factors dissociate from the small ribosomal sub¬ 
unit to make way for the large ribosomal subunit to bind and complete 
ribosomal assembly. Because the initiator tRNA is bound to the P site, 
protein synthesis is ready to begin with the addition of the next charged 
tRNA to the A site (see Figure 7-34). 

The mechanism for selecting a start codon is different in bacteria. Bacterial 
mRNAs have no 5' caps to tell the ribosome where to begin searching for 
the start of translation. Instead, they contain specific ribosome-binding 
sequences, up to six nucleotides long, that are located a few nucleotides 
upstream of the AUGs at which translation is to begin. Unlike a eukaryo¬ 
tic ribosome, a prokaryotic ribosome can readily bind directly to a start 
codon that lies in the interior of an mRNA, as long as a ribosome-binding 
site precedes it by several nucleotides. Such ribosome-binding sequences 
are necessary in bacteria, as prokaryotic mRNAs are often polycistronic— 
that is, they encode several different proteins, each of which is translated 
from the same mRNA molecule (Figure 7-37). In contrast, a eukaryotic 
mRNA usually carries the information for a single protein. 

The end of translation in both prokaryotes and eukaryotes is signaled by 
the presence of one of several codons, called stop codons, in the mRNA 
(see Figure 7-25). The stop codons—UAA, UAG, and UGA—are not recog¬ 
nized by a tRNA and do not specify an amino acid, but instead signal to 
the ribosome to stop translation. Proteins known as release factors bind 
to any stop codon that reaches the A site on the ribosome; this binding 
alters the activity of the peptidyl transferase in the ribosome, causing it to 
catalyze the addition of a water molecule instead of an amino acid to the 
peptidyl-tRNA (Figure 7-38). This reaction frees the carboxyl end of the 
polypeptide chain from its attachment to a tRNA molecule; because this 
is the only attachment that holds the growing polypeptide to the ribos¬ 
ome, the completed protein chain is immediately released. At this point, 
the ribosome also releases the mRNA and dissociates into its two sepᬠ
rate subunits, which can then assemble on another mRNA molecule to 
begin a new round of protein synthesis. 

Figure 7-38 Translation halts at a stop codon. In the final phase of 
protein synthesis, the binding of release factor to an A site bearing 
a stop codon terminates translation of an mRNA molecule. The 
completed polypeptide is released, and the ribosome dissociates 
¡nto its two sepárate subunits. Note that only the 3' end of the mRNA 
molecule is shown here. 

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From RNA to Protein 


We saw in Chapter 4 that many proteins can fold into their three-dimen- 
sional shape spontaneously, and some do so as they are spun out of the 
ribosome. Most proteins, however, require chaperone proteins to help 
them fold correctly in the cell. Chaperones can "steer" proteins along pro- 
ductive folding pathways and prevent them from aggregating inside the 
cell (see Figures 4-9 and 4-10). Newly synthesized proteins are typically 
met by their chaperones as they emerge from the ribosome. 

Proteins Are Made on Polyribosomes 

The synthesis of most protein molecules takes between 20 seconds and 
several minutes. But even during this short period, múltiple ribosomes 
usually bind to each mRNA molecule being translated. If the mRNA is 
being translated efflciently, a new ribosome hops onto the 5' end of the 
mRNA molecule almost as soon as the preceding ribosome has trans¬ 
lated enough of the nucleotide sequence to move out of the way. The 
mRNA molecules being translated are therefore usually found in the form 
of polyribosomes, also known as polysomes. These large cytoplasmic 
assemblies are made up of many ribosomes spaced as cióse as 80 nucle- 
otides apart along a single mRNA molecule (Figure 7-39). With múltiple 
ribosomes working simultaneously on a single mRNA, many more pro¬ 
tein molecules can be made in a given time than would be possible if 
each polypeptide had to be completed before the next could be started. 
Polysomes opérate in both bacteria and eukaryotes, but bacteria can 
speed up the rate of protein synthesis even further. Because bacterial 
mRNA does not need to be processed and is also physically accessible to 
ribosomes while it is being made, ribosomes will typically attach to the 
free end of a bacterial mRNA molecule and start translating it even before 
the transcription of that RNA is complete; these ribosomes follow closely 
behind the RNA polymerase as it moves along DNA. 

Inhibitors of Prokaryotic Protein Synthesis Are Used as 

The ability to transíate mRNAs accurately into proteins is a fundamental 
feature of all life on Earth. Although the ribosome and other molecules 
that carry out this complex task are very similar among organisms, we 

Figure 7-39 Proteins are synthesized on 

polyribosomes. (A) Schematic drawing 
showing how a series of ribosomes can 
simultaneously transíate the same mRNA 
molecule (Movie 7.10). (B) Electron 

micrograph of a polyribosome in the cytosol 


100 nm 


100 nm 

of a eukaryotic cell. (B, courtesy of John 

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From DNAto Protein: How Cells Read the Genome 


1 Antibiotic 

Specific Effect 1 


blocks binding of aminoacyl-tRNA to A site of ribosome 
(step 1 in Figure 7-34) 


prevents the transition from initiation complex to chain 
elongation (see Figure 7-36); also causes miscoding 


blocks the peptidyl transferase reaction on ribosomes 
(step 2 in Figure 7-34) 


blocks the translocation reaction on ribosomes (step 3 in 

Figure 7-34) 


blocks initiation of transcription by binding to RNA 

have seen that there are some subtle differences in the way that bac¬ 
teria and eukaryotes synthesize RNA and proteins. Through a quirk of 
evolution, these differences form the basis of one of the most important 
advances in modern medicine. 

Many of our most effective antibiotics are compounds that act by inhib- 
iting bacterial, but not eukaryotic, RNA and protein synthesis. Some 
of these drugs exploit the small structural and functional differences 
between bacterial and eukaryotic ribosomes, so that they interfere pref- 
erentially with bacterial protein synthesis. These compounds can thus 
be taken in doses high enough to kill bacteria without being toxic to 
humans. Because different antibiotics bind to different regions of the 
bacterial ribosome, these drugs often inhibit different steps in protein 
synthesis. A few of the antibiotics that inhibit bacterial RNA and protein 
synthesis are listed in Table 7-3. 

Many common antibiotics were first isolated from fungí. Fungí and bacte¬ 
ria often occupy the same ecological niches; to gain a competitive edge, 
fungí have evolved, over time, potent toxins that kill bacteria but are 
harmless to themselves. Because fungí and humans are both eukaryotes, 
and are thus more closely related to each other than either is to bacteria 
(see Figure 1-28), we have been able to borrow these weapons to combat 
our own bacterial foes. 

Controlled Protein Breakdown Helps Regúlate the Amount 
of Each Protein in a Cell 

After a protein is released from the ribosome, a cell can control its activ- 
ity and longevity in various ways. The number of copies of a protein in 
a cell depends, like the human population, not only on how quickly new 
individuáis are made but also on how long they survive. So controlling 
the breakdown of proteins into their constituent amino acids helps cells 
regúlate the amount of each particular protein. Proteins vary enormously 
in their life-span. Structural proteins that become part of a relatively sta- 
ble tissue such as bone or muscle may last for months or even years, 
whereas other proteins, such as metabolic enzymes and those that regú¬ 
late cell growth and división (discussed in Chapter 18), last only for days, 
hours, or even seconds. How does the cell control these lifetimes? 

Cells possess specialized pathways that enzymatically break proteins 
down into their constituent amino acids (a process termed proteolysis). 
The enzymes that degrade proteins, first to short peptides and finally to 
individual amino acids, are known collectively as proteases. Proteases 

LibertadDigital \ 2015 

From RNA to Protein 


Figure 7-40 A proteasome degrades 
short-lived and misfolded proteins. The 

structures shown were determined by X-ray 
crystallography. (A) A cut-away view of the 
central cylinder of the proteasome, with 
the active sites of the proteases indicated 
by red dots. (B) The structure of the entire 
proteasome, ¡n which access to the central 
cylinder ( yellow) is regulated by a stopper 
(blue) at each end. (B, adapted from P.C.A 
da Fonseca et al., Mol. Ce//46:54-66, 2012.) 

act by cutting (hydrolyzing) the peptide bonds between amino acids (see 
Panel 2-5, pp. 74-75). One function of proteolytic pathways is to rap- 
idly degrade those proteins whose lifetimes musí be kept short. Another 
is to recognize and remove proteins that are damaged or misfolded. 
Eliminating improperly folded proteins is critical for an organism, as mis¬ 
folded proteins tend to aggregate, and protein aggregates can damage 
cells and even trigger cell death. Eventually, all proteins—even long-lived 
ones—accumulate damage and are degraded by proteolysis. 

In eukaryotic cells, proteins are broken down by large protein machines 
called proteasomes, present in both the cytosol and the nucleus. A pro¬ 
teasome contains a central cylinder formed from proteases whose active 
sites face into an inner chamber. Each end of the cylinder is stoppered by 
a large protein complex formed from at least 10 types of protein subunits 
(Figure 7-40). These protein stoppers bind the proteins destined for deg- 
radation and then—using ATP hydrolysis to fuel this activity—unfold the 
doomed proteins and thread them into the inner chamber of the cylinder. 
Once the proteins are inside, proteases chop them into short peptides, 
which are then jettisoned from either end of the proteasome. Housing 
proteases inside these molecular destruction chambers makes sense, as 
it prevenís the enzymes from running rampant in the cell. 

How do proteasomes select which proteins in the cell should be degraded? 
In eukaryotes, proteasomes act primarily on proteins that have been 
marked for destruction by the covalent attachment of a small protein 
called ubiquitin. Specialized enzymes tag selected proteins with a short 
chain of ubiquitin molecules; these ubiquitylated proteins are then rec- 
ognized, unfolded, and fed into proteasomes by proteins in the stopper 
(Figure 7-41). 

Figure 7-41 Proteins marked by a 
polyubiquitin chain are degraded by the 
proteasome. Proteins ¡n the stopper of a 
proteasome (blue) recognize target proteins 
marked by a specific type of polyubiquitin 
chain. The stopper then unfolds the target 
protein and threads it into the proteasome's 
central cylinder (yellow), which is lined with 
proteases that chop the protein to pieces. 

LibertadDigital \ 2015 

252 CHAPTER 7 From DNA to Protein: How Cells Read the Genome 

Proteins that are meant to be short-lived often contain a short amino 
acid sequence that identifies the protein as one to be ubiquitylated and 
degraded in proteasomes. Damaged or misfolded proteins, as well as 
proteins containing oxidized or otherwise abnormal amino acids, are 
also recognized and degraded by this ubiquitin-dependent proteolytic 
system. The enzymes that add a polyubiquitin chain to such proteins rec- 
ognize signáis that become exposed on these proteins as a result of the 
misfolding or Chemical damage—for example, amino acid sequences or 
conformational motifs that remain buried and inaccessible in the normal 
"healthy" protein. 

There Are Many Steps Between DNA and Protein 

We have seen that many types of Chemical reactions are required to 
produce a protein from the information contained in a gene. The final 
concentration of a protein in a cell therefore depends on the rate at which 
each of the many steps is carried out (Figure 7-42). In addition, many 
proteins—once they leave the ribosome—require further attention before 
they are useful to the cell. Examples of such post-translational modifica- 
tions inelude covalent modification (such as phosphorylation), the binding 
of small-molecule cofactors, or association with other protein subunits, 
which are often needed for a newly synthesized protein to become fully 
functional (Figure 7-43). 

Figure 7-42 Protein production in a 
eukaryotic cell requires many steps. The 

final concentration of each protein depends 
on the rate of each step depicted. Even after 
an mRNA and its corresponding protein have 
been produced, their concentrations can 
be regulated by degradation. Although not 
shown here, the activity of the protein can 
also be regulated by other post-translational 
modifications orthe binding of small 
molecules (see Figure 7-43). 

LibertadDigital \ 2015 


We will see in the next chapter that cells have the ability to change the 
concentrations of most of their proteins according to their needs. In prin¬ 
cipie, all of the steps in Figure 7-42 can be regulated by the cell—and 
many of them, in fact, are. However, as we will see in the next chapter, 
the initiation of transcription is the most common point for a cell to regú¬ 
late the expression of its genes. 

Transcription and translation are universal processes that lie at the heart 
of life. However, when scientists carne to consider how the flow of infor- 
mation from DNA to protein might have originated, they carne to some 
unexpected conclusions. 


The central dogma—that DNA makes RNA that makes protein—presented 
evolutionary biologists with a knotty puzzle: if nucleic acids are required 
to direct the synthesis of proteins, and proteins are required to synthe- 
size nucleic acids, how could this System of interdependent components 
have arisen? One view is that an RNA world existed on Earth before 
cells containing DNA and proteins appeared. According to this hypoth- 
esis, RNA—which today serves largely as an intermedíate between genes 
and proteins—both stored genetic information and catalyzed Chemical 
reactions in primitive cells. Only later in evolutionary time did DNA take 
over as the genetic material and proteins become the major catalysts 
and structural components of cells (Figure 7-44). If this idea is correct, 
then the transition out of the RNA world was never completed; as we 
have seen, RNA still catalyzes several fundamental reactions in modern 
cells. These RNA catalysts—or ribozymes—including those that opérate 
in the ribosome and in the RNA-splicing machinery, can thus be viewed 
as molecular fossils of an earlier world. 

Life Requires Autocatalysis 

The origin of life requires molecules that possess, if only to a small extent, 
one crucial property: the ability to catalyze reactions that lead—directly or 
indirectly—to the production of more molecules like themselves. Catalysts 
with this self-producing property, once they had arisen by chance, would 
divert raw materials from the production of other substances to make 
more of themselves. In this way, one can envisage the gradual develop- 
ment of an increasingly complex Chemical System of organic monomers 
and polymers that function together to generate more molecules of the 
same types, fueled by a supply of simple raw materials in the primitive 
environment on Earth. Such an autocatalytic System would have many 
of the properties we think of as characteristic of living matter: the Sys¬ 
tem would contain a far-from-random selection of interacting molecules; 
it would tend to reproduce itself; it would compete with other Systems 
dependent on the same raw materials; and, if deprived of its raw mat¬ 
erials or maintained at a temperature that upset the balance of reaction 
rates, it would decay toward Chemical equilibrium and "die." 

RNA and the Origins of Life 

nascent polypeptide chain 




mature functlonal protein 

Figure 7-43 Many proteins require 
various modifications to become fully 
functional. To be useful to the cell, a 
completed polypeptide must fold correctly 
into its three-dimensional conformation 
and then bind any required cofactors (red) 
and protein partners—all via noncovalent 
bonding. Many proteins also require one 
or more covalent modifications to become 
active—orto be recruited to specific 
membranes or organelles (not shown). 
Although phosphorylation and glycosylation 
are the most common, more than 100 types 
of covalent modifications of proteins are 

Big Bang 

system first cells first 

formed with DNA mammals 

5 V 



Figure 7-44 An RNA world may have 
existed before modern cells with DNA 
and proteins evolved. 

LibertadDigital \ 2015 



From DNAto Protein: How Cells Read the Genome 

But what molecules could have had such autocatalytic properties? In 
present-day living cells, the most versatile catalysts are proteins, which 
are able to adopt diverse three-dimensional forms that bristle with chem- 
ically reactive sites on their surface. However, there is no known way in 
which a protein can reproduce itself directly. RNA molecules, by contrast, 
could—at least, in principie—catalyze their own synthesis. 

RNA Can Both Store Information and Catalyze Chemical 

We have seen that complementary base-pairing enables one nucleic acid 
to act as a témplate for the formation of another. Thus a single strand of 
RNA or DNA can specify the sequence of a complementary polynucle- 
otide, which, in tum, can specify the sequence of the original molecule, 
allowing the original nucleic acid to be replicated (Figure 7-45). Such 
complementary templating mechanisms lie at the heart of both DNA rep- 
lication and transcription in modern-day cells. 

But the efflcient synthesis of polynucleotides by such complementary 
templating mechanisms also requires catalysts to promote the polymeri- 
zation reaction: without catalysts, polymer formation is slow, error-prone, 
and inefflcient. Today, nucleotide polymerization is catalyzed by protein 
enzymes—such as DNA and RNA polymerases. But how could this reac¬ 
tion be catalyzed before proteins with the appropriate catalytic ability 
existed? The beginnings of an answer were obtained in 1982, when it 
was discovered that RNA molecules themselves can act as catalysts. The 
unique potential of RNA molecules to act both as information carriers 
and as catalysts is thought to have enabled them to have a central role 
in the origin of life. 

In present-day cells, RNA is synthesized as a single-stranded molecule, 
and we have seen that complementary base-pairing can occur between 
nucleotides in the same chain. This base-pairing, along with noncon- 
ventional hydrogen bonds, can cause each RNA molecule to fold up in 
a unique way that is determined by its nucleotide sequence (see Figure 
7-5). Such associations produce complex three-dimensional shapes. 

As we discuss in Chapter 4, protein enzymes are able to catalyze bio- 
chemical reactions because they have surfaces with unique contours 
and Chemical properties. In the same way, RNA molecules, with their 
unique folded shapes, can serve as catalysts (Figure 7-46). RNAs do not 
have the same structural and functional diversity as do protein enzymes; 
they are, after all, built from only four different subunits. Nonetheless, 
ribozymes can catalyze many types of Chemical reactions. Most of the 
ribozymes that have been studied were constructed in the laboratory and 
selected for their catalytic activity in a test tube (Table 7-4), as relatively 
few catalytic RNAs exist in present-day cells. But the processes in which 
catalytic RNAs still seem to have major roles inelude some of the most 

Figure 7-45 An RNA molecule can in 
principie guide the formation of an 
exact copy of itself. In the first step, the 
original RNA molecule acts as a témplate to 
form an RNA molecule of complementary 
sequence. In the second step, this 
complementary RNA molecule itself acts 
as a témplate to form an RNA molecule of 
the original sequence. Since each témplate 
molecule can produce many copies of the 
complementary strand, these reactions can 
result in the amplification of the original 



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RNA and the Origins of Life 




1 Activity 

1 Ribozymes 1 

Peptlde bond formation in protein 

ribosomal RNA 

DNA ligatlon 

¡n v/tro selected RNA 

RNA spllclng 

self-spliclng RNAs, small nuclear RNAs 

RNA polymerizatlon 

¡n v/tro selected RNA 

RNA phosphorylatlon 

in v/tro selected RNA 

RNA amlnoacylation 

in v/tro selected RNA 

RNA alkylatlon 

in v/tro selected RNA 

C-C bond rotatlon (¡somerizatlon) 

in v/tro selected RNA 

fundamental steps in the expression of genetic information—especially 
those steps where RNA molecules themselves are spliced or translated 
into protein. 

RNA, therefore, has all the properties required of a molecule that could 
catalyze its own synthesis (Figure 7-47). Although self-replicating Sys¬ 
tems of RNA molecules have not been found in nature, scientists appear 
to be well on the way to constructing them in the laboratory. Although 
this demonstration would not prove that self-replicating RNA molecules 
were essential to the origin of life on Earth, it would establish that such 
a scenario is possible. 

RNA Is Thought to Predate DNA ¡n Evolution 

The first cells on Earth would presumably have been much less com- 
plex and less efficient in reproducing themselves than even the simplest 
present-day cells. They would have consisted of little more than a simple 
membrane enclosing a set of self-replicating molecules and a few other 
components required to provide the materials and energy for this auto- 
catalytic replication. If the evolutionaiy role for RNA proposed above is 
correct, these earliest cells would also have differed fundamentally from 
the cells we know today in having their hereditary information stored in 
RNA rather than DNA. 

Evidence that RNA aróse before DNA in evolution can be found in the 
Chemical differences between them. Ribose (see Figure 7-3A), like 





Figure 7-46 A ribozyme ¡s an RNA 
molecule that possesses catalytic activity. 

The RNA molecule shown catalyzes the 
cleavage of a second RNA at a speclflc 
site. Similar rlbozymes are found embedded 
¡n large RNA genomes—called virolds— 
that infect plants, where the cleavage 
reactlon Is one step ¡n the replication 
of the virold. (Adapted from T.R. Cech and 
O.C. Uhlenbeck, Nature 372:39-40, 1994. 
Wlth permisslon from Macmlllan 
Publlshers Ltd.) 

Figure 7-47 Could an RNA molecule catalyze ¡ts 
own synthesis? This hypothetlcal process would 
requlre that the RNA catalyze both steps shown ¡n 
Figure 7-45. The red rays represent the active site 
of this ribozyme. 

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CHAPTER 7 From DNAto Protein: How Cells Read the Genome 



Figure 7-48 RNA may have preceded 
DNA and proteins in evolution. According 
to this hypothesis, RNA molecules provided 
genetic, structural, and catalytic functions in 
the earliest cells. DNA ¡s nowthe repository 
of genetic information, and proteins carry 
out almost all catalysis in cells. RNA now 
functions mainly as a go-between in protein 
synthesis, while remaining a catalyst for 
a few crucial reactions (¡ncluding protein 


Discuss the following: "During the 
evolution of life on Earth, RNA lost 
its glorious position as the first self- 
replicating catalyst. Its role now is as 
a mere messenger in the information 
flow from DNA to protein." 

glucose and other simple carbohydrates, is readily formed from formal- 
dehyde (HCHO), which is one of the principal products of experiments 
simulating conditions on the primitive Earth. The sugar deoxyribose is 
harder to make, and in present-day cells it is produced from ribose in 
a reaction catalyzed by a protein enzyme, suggesting that ribose pre¬ 
dates deoxyribose in cells. Presumably, DNA appeared on the scene after 
RNA, and then proved more suited than RNA as a permanent repository 
of genetic information. In particular, the deoxyribose in its sugar-phos- 
phate backbone makes chains of DNA chemically much more stable than 
chains of RNA, so that greater lengths of DNA can be maintained without 

The other differences between RNA and DNA—the double-helical struc- 
ture of DNA and the use of thymine rather than uracil—further enhance 
DNA stability by making the molecule easier to repair. We saw in Chapter 
6 that a damaged nucleotide on one strand of the double helix can be 
repaired by using the other strand as a témplate. Furthermore, deamina- 
tion, one of the most common unwanted Chemical changes occurring in 
polynucleotides, is easier to detect and repair in DNA than in RNA (see 
Figure 6-23). This is because the product of the deamination of cytosine 
is, by chance, uracil, which already exists in RNA, so that such damage 
would be impossible for repair enzymes to detect in an RNA molecule. 
However, in DNA, which has thymine rather than uracil, any uracil pro¬ 
duced by the accidental deamination of cytosine is easily detected and 

Taken together, the evidence we have discussed supports the idea that 
RNA—with its ability to provide genetic, structural, and catalytic func¬ 
tions—preceded DNA in evolution. As cells more closely resembling 
present-day cells appeared, it is believed that many of the functions orig- 
inally performed by RNA were taken over by DNA and proteins: DNA 
took over the primary genetic function, and proteins became the major 
catalysts, while RNA remained primarily as the intermediary connecting 
the two (Figure 7-48). With the advent of DNA, cells were able to become 
more complex, for they could then carry and transmit more genetic 
information than could be stably maintained by RNA alone. Because of 
the greater Chemical complexity of proteins and the variety of Chemi¬ 
cal reactions they can catalyze, the shift (albeit incomplete) from RNA 
to proteins also provided a much richer source of structural components 
and enzymes. This enabled cells to evolve the great diversity of structure 
and function that we see in life today. 


• The flow of genetic information in all living cells is DNA —► RNA —► 
protein. The conversión of the genetic instructions in DNA into RNAs 
and proteins is termed gene expression. 

• To express the genetic information carried in DNA, the nucleotide 
sequence of a gene is first transcribed into RNA. Transcription is 
catalyzed by the enzyme RNA polymerase, which uses nucleotide 
sequences in the DNA molecule to determine which strand to use as 
a témplate, and where to start and stop transcribing. 

• RNA differs in several respects from DNA. It contains the sugar ribose 
instead of deoxyribose and the base uracil (U) instead of thymine (T). 
RNAs in cells are synthesized as single-stranded molecules, which 
often fold up into complex three-dimensional shapes. 

• Cells make several functional types of RNAs, including messenger 
RNAs (mRNAs), which carry the instructions for making proteins; 
ribosomal RNAs (rRNAs), which are the crucial components of 

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ribosomes; and transfer RNAs (tRNAs), which act as adaptor mol- 
ecules in protein synthesis. 

To begin transcription, RNA polymerase binds to specific DNA sites 
called promoters that lie immediately upstream of genes. To initiate 
transcription, eukaiyotic RNA polymerases require the assembly of 
a complex of general transcription factors at the promoter, whereas 
bacterial RNA polymerase requires only an additional subunit, called 
sigma factor. 

Most protein-coding genes in eukaiyotic cells are composed of a 
number of coding regions, called exons, interspersed with larger 
noncoding regions, called introns. When a eukaiyotic gene is tran- 
scribed from DNA into RNA, both the exons and introns are copied. 
Introns are removed from the RNA transcripts in the nucleus by RNA 
splicing, a reaction catalyzed by small ribonucleoprotein complexes 
known as snRNPs. Splicing removes the introns from the RNA and 
joins together the exons—often in a variety of combinations, allowing 
múltiple proteins to be produced from the same gene. 

Eukaiyotic pre-mRNAs go through several additional RNA Process¬ 
ing steps before they leave the nucleus as mRNAs, including 5' RNA 
capping and 3' polyadenylation. These reactions, along with splicing, 
take place as the pre-mRNA is being transcribed. 

Translation of the nucleotide sequence of an mRNA into a protein 
takes place in the cytoplasm on large ribonucleoprotein assemblies 
called ribosomes. As the mRNA moves through the ribosome, its 
message is translated into protein. 

The nucleotide sequence in mRNA is read in sets of three nucleotides 
called codons; each codon corresponds to one amino acid. 

The correspondence between amino acids and codons is specified 
by the genetic code. The possible combinations of the 4 different 
nucleotides in RNA give 64 different codons in the genetic code. Most 
amino acids are specified by more than one codon. 
tRNAs act as adaptor molecules in protein synthesis. Enzymes called 
aminoacyl-tRNA synthetases covalently link amino acids to their 
appropriate tRNAs. Each tRNA contains a sequence of three nucle¬ 
otides, the anticodon, which recognizes a codon in an mRNA through 
complementaiy base-pairing. 

Protein synthesis begins when a ribosome assembles at an initia- 
tion codon (AUG) in an mRNA molecule, a process that depends on 
proteins called translation initiation factors. The completed protein 
chain is released from the ribosome when a stop codon (UAA, UAG, 
or UGA) in the mRNA is reached. 

The stepwise linking of amino acids into a polypeptide chain is cata¬ 
lyzed by an rRNA molecule in the large ribosomal subunit, which thus 
acts as a ribozyme. 

The concentration of a protein in a cell depends on the rate at 
which the mRNA and protein are synthesized and degraded. Protein 
degradation in the cytosol and nucleus occurs inside large protein 
complexes called proteasomes. 

From our knowledge of present-day organisms and the molecules 
they contain, it seems likely that life on Earth began with the evolu- 
tion of RNA molecules that could catalyze their own replication. 

It has been proposed that RNA served as both the genome and the 
catalysts in the first cells, before DNA replaced RNA as a more stable 
molecule for storing genetic information, and proteins replaced RNAs 
as the major catalytic and structural components. RNA catalysts in 
modem cells are thought to provide a glimpse into an ancient, RNA- 
based world. 

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CHAPTER 7 From DNAto Protein: How Cells Read the Genome 


alternative splicing 

messenger RNA (mRNA) 

RNA polymerase 

aminoacyl-tRNA synthetase 


RNA processing 



RNA splicing 



RNA transcript 



RNA world 


reading frame 

small nuclear RNA (snRNA) 

gene expression 

ribosomal RNA (rRNA) 


general transcription factors 



genetic code 


transfer RNA (tRNA) 

initiator tRNA 




RNA capping 

translation initiation factor 



Which of the following statements are corred? Explain your 

A. An individual ribosome can make only one type of 

B. All mRNAs fold into particular three-dimensional 
strudures that are required for their translation. 

C. The large and small subunits of an individual ribosome 
always stay together and never exchange partners. 

D. Ribosomes are cytoplasmic organelles that are 
encapsulated by a single membrane. 

E. Because the two strands of DNA are complementary, 
the mRNA of a given gene can be synthesized using either 
strand as a témplate. 

F. An mRNA may contain the sequence 

G. The amount of a protein present in a cell depends on 
its rate of synthesis, its catalytic adivity, and its rate of 


The Lacheinmal protein is a hypothetical protein that causes 
people to smile more often. It is inadive in many chronically 
unhappy people. The mRNA isolated from a number of 
different unhappy individuáis in the same family was found 
to lack an internal stretch of 173 nucleotides that is present 
in the Lacheinmal mRNA isolated from happy members of 
the same family. The DNA sequences of the Lacheinmal 
genes from the happy and unhappy family members were 
determined and compared. They differed by a single 
nudeotide substitution, which lay in an intron. What can you 
say about the molecular basis of unhappiness in this family? 

(Hints: [1] Can you hypothesize a molecular mechanism by 
which a single nudeotide substitution in a gene could cause 
the observed deletion in the mRNA? Note that the deletion 
is infernal to the mRNA. [2] Assuming the 173-base-pair 
deletion removes coding sequences from the Lacheinmal 
mRNA, how would the Lacheinmal protein differ between 
the happy and unhappy people?) 


Use the genetic code shown in Figure 7-25 to identify which 
of the following nudeotide sequences would code for the 
polypeptide sequence arginine-glycine-aspartate: 

1. 5'-AGA-GGA-GAU-3' 

2. 5'-ACA-CCC-ACU-3' 

3. 5'-GGG-AAA-UUU-3' 

4. 5'-CGG-GGU-GAC-3' 


"The bonds that form between the anticodon of a tRNA 
molecule and the three nucleotides of a codon in mRNA are 

_." Complete this sentence with each of the following 

options and explain why each of the resulting statements is 
correct or ¡ncorrect. 

A. Covalent bonds formed by GTP hydrolysis 

B. Hydrogen bonds that form when the tRNA is at the 
A site 

C. Broken by the translocation of the ribosome along the 


List the ordinary, dictionary definitions of the terms 
replication, transcription, and translation. By their side, list 
the special meaning each term has when applied to the 
living cell. 


In an alien world, the genetic code is written in pairs of 
nucleotides. How many amino acids could such a code 
specify? In a different world, a triplet code is used, but the 
sequence of nucleotides is not important; it only matters 
which nucleotides are present. How many amino acids could 
this code specify? Would you expect to encounter any 
problems translating these codes? 

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One remarkable feature of the genetic code is that amino 
acids with similar Chemical properties often have similar 
codons. Thus codons with U or C as the second nucleotide 
tend to specify hydrophobic amino acids. Can you suggest 
a possible explanation for this phenomenon in terms of the 
early evolution of the protein-synthesis machinery? 


A mutation in DNA generates a UGA stop codon in the 
middle of the mRNA coding for a particular protein. 

A second mutation in the cell's DNA leads to a single 
nucleotide change in a tRNA that allows the correct 
translation of the protein; that is, the second mutation 
"suppresses" the defect caused by the first. The altered 
tRNA translates the UGA as tryptophan. What nucleotide 
change has probably occurred in the mutant tRNA 
molecule? What consequences would the presence of such 
a mutant tRNA have for the translation of the normal genes 
in this cell? 


The charging of a tRNA with an amino acid can be 
represented by the following equation: 
amino acid + tRNA + ATP —> aminoacyl-tRNA + AMP + PP¡ 
where PP¡ is pyrophosphate (see Figure 3-40). In the 
aminoacyl-tRNA, the amino acid and tRNA are linked with 
a high-energy covalent bond; a large portion of the energy 
derived from the hydrolysis of ATP is thus stored in this 
bond and is available to drive peptide bond formation at the 
later stages of protein synthesis. The free-energy change of 
the charging reaction shown in the equation is cióse to zero 
and therefore would not be expected to favor attachment 
of the amino acid to tRNA. Can you suggest a further step 
that could drive the reaction to completion? 


A. The average molecular weight of a protein in the cell is 
about 30,000 daltons. A few proteins, however, are much 
larger. The largest known polypeptide chain made by any 
cell is a protein called titin (made by mammalian muscle 
cells), and it has a molecular weight of 3,000,000 daltons. 
Estímate how long it will take a muscle cell to transíate 

an mRNA coding for titin (assume the average molecular 
weight of an amino acid to be 120, and a translation rate of 
two amino acids per second for eukaryotic cells). 

B. Protein synthesis is very accurate: for every 10,000 
amino acids joined together, only one mistake is made. 

What is the fraction of average-sized protein molecules and 
of titin molecules that are synthesized without any errors? 
(Hint: the probability P of obtaining an error-free protein is 
given by P = (1 - E) n , where E is the error frequency and n 
the number of amino acids.) 

C. The molecular weight of all eukaryotic ribosomal 
proteins combined is about 2.5 X 10 6 daltons. Would it be 
advantageous to synthesize them as a single protein? 

D. Transcription occurs at a rate of about 30 nudeotides 
per second. Is it possible to calcúlate the time required to 
synthesize a titin mRNA from the information given here? 

Chapter 7 End-of-Chapter Questions 259 


Which of the following types of mutations would be 
predicted to harm an organism? Explain your answers. 

A. Insertion of a single nucleotide near the end of the 
coding sequence. 

B. Removal of a single nucleotide near the beginning of the 
coding sequence. 

C. Deletion of three consecutive nudeotides in the middle 
of the coding sequence. 

D. Deletion of four consecutive nudeotides in the middle of 
the coding sequence. 

E. Substitution of one nucleotide for another in the middle 
of the coding sequence. 

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left intentionally blank 

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Control of Gene Expression 

An organism's DNA encodes all of the RNA and protein molecules that are 
needed to make its cells. Yet a complete description of the DNA sequence 
of an organism—be it the few million nucleotides of a bacterium or the 
few billion nucleotides in each human cell—does not enable us to recon- 
struct that organism any more than a list of all the English words in a 
dictionary enables us to reconstruct a play by Shakespeare. We need to 
know how the elements in the DNA sequence or the words on a list work 
together to make the masterpiece. 

For cells, the question involves gene expression. Even the simplest single- 
celled bacterium can use its genes selectively—for example, switching 
genes on and off to make the enzymes needed to digest whatever food 
sources are available. In multicellular plants and animáis, however, gene 
expression is under much more elabórate control. Over the course of 
embryonic development, a fertilized egg cell gives rise to many cell types 
that differ dramatically in both structure and function. The differences 
between an information-processing nerve cell and an infection-fighting 
white blood cell, for example, are so extreme that it is difficult to imagine 
that the two cells contain the same DNA (Figure 8-1). For this reason, 
and because cells in an adult organism rarely lose their distinctive char- 
acteristics, biologists originally suspected that certain genes might be 
selectively lost when a cell becomes specialized. We now know, how¬ 
ever, that nearly all the cells of a multicellular organism contain the 
same genome. Cell differentiation is instead achieved by changes in gene 

In mammals, hundreds of different cell types carry out a range of spe¬ 
cialized functions that depend upon genes that are switched on in that 






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CHAPTER 8 Control of Gene Expression 

Figure 8-1 A neuron and a liver cell share the same genome. 

The long branches of this neuron from the retina enable it to receive 
electrical signáis from many other neurons and carry them to many 
neighboring neurons. The ¡¡ver cell, which is drawn to the same scale, 
¡s involved in many metabolic processes, including digestión and the 
detoxification of alcohol and other drugs. Both of these mammalian 
cells contain the same genome, butthey express many different RNAs 
and proteins. (Neuron adapted from S. Ramón y Cajal, Histologie 
du Systéme Nerveux de l'Homme et de Vertébrés, 1909-1911. París: 
Maloine; reprinted, Madrid: C.S.I.C., 1972.) 

cell type but not in most others: for example, the p cells of the páncreas 
make the protein hormone insulin, while the a cells of the páncreas make 
the hormone glucagon; the B lymphocytes of the immune System make 
antibodies, while developing red blood cells make the oxygen-transport 
protein hemoglobin. The differences between a neuron, a white blood 
cell, a pancreatic p cell, and a red blood cell depend upon the precise 
control of gene expression. A typical differentiated cell expresses only 
about half the genes in its total repertoire. 

In this chapter, we discuss the main ways in which gene expression is 
regulated, with a focus on those genes that encode proteins as their 
final product. Although some of these control mechanisms apply to both 
eukaryotes and prokaryotes, eukaiyotic cells—with their more complex 
chromosomal structure—have some ways of controlling gene expression 
that are not available to bacteria. 


Gene expression is a complex process by which cells selectively direct 
the synthesis of the many thousands of proteins and RNAs encoded in 
their genome. But how do cells coordínate and control such an intricate 
process—and how does an individual cell specify which of its genes to 
express? This decisión is an especially important problem for animáis 
because, as they develop, their cells become highly specialized, ulti- 
mately producing an array of muscle, nerve, and blood cells, along with 
the hundreds of other cell types seen in the adult. Such cell differentia- 
tion arises because cells make and accumulate different sets of RNA and 
protein molecules: that is, they express different genes. 

The Different Cell Types of a Multicellular Organism 
Contain the Same DNA 

The evidence that cells have the ability to change which genes they 
express without altering the nucleotide sequence of their DNA comes 
from experiments in which the genome from a differentiated cell is made 
to direct the development of a complete organism. If the chromosomes of 
the differentiated cell were altered irreversibly during development, they 
would not be able to accomplish this feat. 

Consider, for example, an experiment in which the nucleus is taken from 
a skin cell in an adult frog and injected into a frog egg from which the 
nucleus has been removed. In at least some cases, that doctored egg 
will develop into a normal tadpole (Figure 8-2). Thus, the transplanted 
skin-cell nucleus cannot have lost any critical DNA sequences. Nuclear 
transplantation experiments carried out with differentiated cells taken 
from adult mammals—including sheep, cows, pigs, goats, and mice— 
have shown similar results. And in plants, individual cells removed from 
a carrot, for example, can regenérate an entire adult carrot plant. These 
experiments all show that the DNA in specialized cell types of multicel¬ 
lular organisms still contains the entire set of instructions needed to form 

LibertadDigital \ 2015 

An OverView of Gene Expression 



unfertilized egg nudeus destroyed 

by UV light 

section proliferating separated single 

ofcarrot cell mass cells in rich cell 


normal embryo 

done of young 

dividing embryo 


young carrot 


embryo placed in 
foster mother 

Figure 8-2 Differentiated cells contain all the genetic ¡nstructions necessary to direct the formation of a 
complete organism. (A) The nucleus of a skin cell from an adult frog transplanted into an egg whose nucleus has 
been destroyed can give rise to an entire tadpole. The broken arrow indicates that to give the transplanted genome 
time to adjust to an embryonic environment, a further transfer step is required in which one of the nuclei ¡s taken 
from the early embryo that begins to develop and is put back into a second enucleated egg. (B) In many types of 
plants, differentiated cells retain the ability to "de-differentiate," so that a single cell can proliferate to form a clone 
of progeny cells that later give rise to an entire plant. (C) A nucleus removed from a differentiated cell from an adult 
cow can be introduced into an enucleated egg from a different cow to give rise to a calf. Different calves produced 
from the same differentiated cell donor are all clones of the donor and are therefore genetically ¡dentical. 

(A, modified from J.B. Gurdon, Sci. Am. 219:24-35, 1968, with permission from the Estate of Bunji Tagawa.) 

a whole organism. The various cell types of an organism therefore differ 
not because they contain different genes, but because they express them 

Different Cell Types Produce Different Sets of Proteins 

The extent of the differences in gene expression between different cell 
types may be roughly gauged by comparing the protein composition of 
cells in liver, heart, brain, and so on. In the past, such analysis was per- 
formed by two-dimensional gel electrophoresis (see Panel 4-5, p. 167). 
Nowadays, the total protein content of a cell can be rapidly analyzed by 

LibertadDigital \ 2015 

264 CHAPTER 8 Control of Gene Expression 

a method called mass spectrometry (see Figure 4-49). This technique is 
much more sensitive than electrophoresis and it enables the detection of 
even proteins that are produced in minor quantities. 

Both techniques reveal that many proteins are common to all the cells of 
a multicellular organism. These housekeeping proteins inelude, for exam- 
ple, the structural proteins of chromosomes, RNA polymerases, DNA 
repair enzymes, ribosomal proteins, enzymes involved in glycolysis and 
other basic metabolic processes, and many of the proteins that form the 
cytoskeleton. In addition, each different cell type also produces special- 
ized proteins that are responsible for the cell's distinctive properties. In 
mammals, for example, hemoglobin is made almost exclusively in devel- 
oping red blood cells. 

Gene expression can also be studied by cataloging a cell's RNAs, includ- 
ing the mRNAs that encode protein. The most comprehensive methods for 
such analyses involve determining the nucleotide sequence of every RNA 
molecule made by the cell, an approach that can also reveal their rela- 
tive abundance. Estimates of the number of different mRNA sequences in 
human cells suggest that, at any one time, a typical differentiated human 
cell expresses perhaps 5000-15,000 protein-coding genes from a total of 
about 21,000. It is the expression of a different collection of genes in each 
cell type that causes the large variations seen in the size, shape, behavior, 
and function of differentiated cells. 

A Cell Can Change the Expression of Its Genes in 
Response to External Signáis 

The specialized cells in a multicellular organism are capable of alter- 
ing their pattems of gene expression in response to extracellular cues. 
For example, if a liver cell is exposed to the steroid hormone cortisol, 
the production of several proteins is dramatically increased. Released 
by the adrenal gland during periods of starvation, intense exercise, or 
prolonged stress, cortisol signáis liver cells to boost the production of 
glucose from amino acids and other small molecules. The set of pro¬ 
teins whose production is induced by cortisol ineludes enzymes such 
as tyrosine aminotransferase, which helps convert tyrosine to glucose. 
When the hormone is no longer present, the production of these proteins 
returns to its resting level. 

Other cell types respond to cortisol differently. In fat cells, for example, 
the production of tyrosine aminotransferase is reduced, while some other 
cell types do not respond to cortisol at all. The fact that different cell 
types often respond in different ways to the same extracellular signal 
contributes to the specialization that gives each cell type its distinctive 

Gene Expression Can Be Regulated at Various Steps from 
DNA to RNA to Protein 

If differences among the various cell types of an organism depend on 
the particular genes that the cells express, at what level is the control 
of gene expression exercised? As we saw in the last chapter, there are 
many steps in the pathway leading from DNA to protein, and all of them 
can in principie be regulated. Thus a cell can control the proteins it con- 
tains by (1) controlling when and how often a given gene is transcribed, 

(2) controlling how an RNA transcript is spliced or otherwise processed, 

(3) selecting which mRNAs are exported from the nucleus to the cytosol, 

(4) regulating how quickly certain mRNA molecules are degraded, 

(5) selecting which mRNAs are translated into protein by ribosomes, or 

LibertadDigital \ 2015 

How Transcriptional Switches Work 265 

Figure 8-3 Gene expression 
¡n eukaryotic cells can be 
controlled at various steps. 

Examples of regulation at 
each of these steps are 
known, although for most 
genes the main site of control 
is step 1—transcription of a 
DNA sequence ¡nto RNA. 

(6) regulating how rapidly specific proteins are destroyed after they have 
been made; in addition, the activity of individual proteins can be further 
regulated in a variety of ways. These steps are illustrated in Figure 8-3. 
Gene expression can be regulated at each of these steps. For most genes, 
however, the control of transcription (step number 1 in Figure 8-3) is 
paramount. This makes sense because only transcriptional control can 
ensure that no unnecessary intermediates are synthesized. So it is the 
regulation of transcription—and the DNA and protein components that 
determine which genes a cell transcribes into RNA—that we address flrst. 


Until 50 years ago, the idea that genes could be switched on and off was 
revolutionaiy. This concept was a major advance, and it carne originally 
from studies of how E. coli bacteria adapt to changes in the composition 
of their growth médium. Many of the same principies apply to eukaryotic 
cells. However, the enormous complexity of gene regulation in higher 
organisms, combined with the packaging of their DNA into chromatin, 
creates special challenges and some novel opportunities for control—as 
we will see. We begin with a discussion of the transcription regulators, 
proteins that bind to DNA and control gene transcription. 

Transcription Regulators Bind to Regulatory DNA 

Control of transcription is usually exerted at the step at which the proc- 
ess is initiated. In Chapter 7, we saw that the promoter región of a gene 
binds the enzyme RNA polymerase and correctly orients the enzyme to 
begin its task of making an RNA copy of the gene. The promoters of both 
bacterial and eukaryotic genes inelude a transcription initiation site, where 
RNA synthesis begins, plus a sequence of approximately 50 nucleotide 
pairs that extends upstream from the initiation site (if one likens the 
direction of transcription to the flow of a river). This upstream región 
contains sites that are required for the RNA polymerase to recognize the 
promoter, although they do not bind to RNA polymerase directly. Instead, 
these sequences contain recognition sites for proteins that associate with 
the active polymerase—sigma factor in bacteria (see Figure 7-9) or the 
general transcription factors in eukaryotes (see Figure 7-12). 

In addition to the promoter, nearly all genes, whether bacterial or eukary- 
otic, have regulatory DNA sequences that are used to switch the gene 
on or off. Some regulatory DNA sequences are as short as 10 nucleotide 
pairs and act as simple switches that respond to a single signal; such 
simple regulatory switches predomínate in bacteria. Other regulatory 
DNA sequences, especially those in eukaryotes, are veiy long (some- 
times spanning more than 10,000 nucleotide pairs) and act as molecular 

LibertadDigital \ 2015 


CHAPTER 8 Control of Gene Expression 

Figure 8-4 A transcription regulator interacts with the major groove of a DNA double helix. (A) This regulator 
recognizes DNA via three a hélices, shown as numbered cylinders, which allow the protein to fit into the major 
groove and form tight associations with the base pairs ¡n a short stretch of DNA. This particular structural motif, 
called a homeodomain, is found ¡n many eukaryotic DNA-binding proteins (Movie 8.1). (B) Most of the contacts with 
the DNA bases are made by helix 3 (red), which is shown here end-on. The protein interacts with the edges of the 
nucleoides without disrupting the hydrogen bonds that hold the base pairs together. (C) An asparagine residue 
from helix 3 forms two hydrogen bonds with the adenine in an A-T base pair. The view is end-on looking down the 
DNA double helix, and the protein contacts the base pair from the major groove side. For simplicity, only one amino 
acid-base contact is shown; in reality, transcription regulators form hydrogen bonds (as shown here), ionic bonds, 
and hydrophobic interactions with individual bases in the major groove. Typically, the protein-DNA ¡nterface would 
consist of 10-20 such contacts, each involving a different amino acid and each contributing to the overall strength of 
the protein-DNA interaction. 

microprocessors, integrating information from a variety of signáis into a 
command that dictates how often transcription of the gene is initiated. 
Regulatory DNA sequences do not work by themselves. To have any 
effect, these sequences must be recognized by proteins called tran¬ 
scription regulators. It is the binding of a transcription regulator to a 
regulatory DNA sequence that acts as the switch to control transcription. 
The simplest bacterium produces several hundred different transcrip¬ 
tion regulators, each of which recognizes a different DNA sequence and 
thereby regulates a distinct set of genes. Humans make many more—sev¬ 
eral thousand—indicating the importance and complexity of this form of 
gene regulation in the development and function of a complex organism. 
Proteins that recognize a specific nucleotide sequence do so because 
the surface of the protein fits tightly against the surface features of the 
DNA double helix in that región. Because these surface features will vary 
depending on the nucleotide sequence, different DNA-binding proteins 
will recognize different nucleotide sequences. In most cases, the protein 
inserts into the major groove of the DNA helix and makes a series of 
intímate molecular contacts with the nucleotide pairs within the groove 
(Figure 8-4). Although each individual contact is weak, the 10 to 20 con¬ 
tacts that are typically formed at the protein-DNA interface combine to 
ensure that the interaction is both highly specific and very strong; indeed, 
protein-DNA interactions are among the tightest and most specific 
molecular interactions known in biology. 

Many transcription regulators bind to the DNA helix as dimers (Figure 
8-5). Such dimerization roughly doubles the area of contact with the 
DNA, thereby greatly increasing the strength and specificity of the pro¬ 
tein-DNA interaction. 

LibertadDigital \ 2015 

How Transcriptional Switches Work 267 

Transcriptional Switches Allow Cells to Respond to 
Changes in Their Environment 

The simplest and best understood examples of gene regulation occur in 
bacteria and in the viruses that infect them. The genome of the bacterium 
E. coli consists of a single circular DNA molecule of about 4.6 x 10 6 nucle- 
otide pairs. This DNA encodes approximately 4300 proteins, although 
only a fraction of these are made at any one time. Bacteria regúlate the 
expression of many of their genes according to the food sources that are 
available in the environment. For example, in E. coli, five genes code for 
enzymes that manufacture the amino acid tryptophan. These genes are 
arranged in a cluster on the chromosome and are transcribed from a sin¬ 
gle promoter as one long mRNA molecule; such coordinately transcribed 
clusters are called operons (Figure 8-6). Although operons are common 
in bacteria, they are rare in eukaryotes, where genes are transcribed and 
regulated individually (see Figure 7-2). 

When tiyptophan concentrations are low, the operon is transcribed; 
the resulting mRNA is translated to produce a full set of biosynthetic 
enzymes, which work in tándem to synthesize tiyptophan. When tryp¬ 
tophan is abundant, however—for example, when the bacterium is in 
the gut of a mammal that has just eaten a protein-rich meal—the amino 
acid is imported into the cell and shuts down production of the enzymes, 
which are no longer needed. 

We now understand in considerable detail how this repression of the 
tiyptophan operon comes about. Within the operon's promoter is a short 
DNA sequence, called the operator (see Figure 8-6), that is recognized 
by a transcription regulator. When this regulator binds to the operator, it 
blocks access of RNA polymerase to the promoter, preventing transcrip¬ 
tion of the operon and production of the tryptophan-producing enzymes. 
The transcription regulator is known as the tiyptophan repressor, and it is 
controlled in an ingenious way: the repressor can bind to DNA only if it 
has also bound several molecules of tiyptophan (Figure 8-7). 

The tiyptophan repressor is an allosteric protein (see Figure 4-41): the 
binding of tiyptophan causes a subtle change in its three-dimensional 
structure so that the protein can bind to the operator sequence. When 
the concentration of free tiyptophan in the bacterium drops, the repres¬ 
sor no longer binds to DNA, and the tryptophan operon is transcribed. 
The repressor is thus a simple device that switches production of a set of 
biosynthetic enzymes on and off according to the availability of the end 
product of the pathway that the enzymes catalyze. 

The tiyptophan repressor protein itself is always present in the cell. The 
gene that encodes it is continuously transcribed at a low level, so that a 
small amount of the repressor protein is always being made. Thus the 
bacterium can respond very rapidly to a rise in tryptophan concentration. 


I E_D_C B 


E. coli chromosome 

( 1(11 

r p * ♦ » 

series of enzymes required for tryptophan biosynthesis 

Figure 8-5 Many transcription regulators 
bind to DNA as dimers. This transcription 
regulator contains a leucine zipper motif, 
which is formed by two a hélices, each 
contributed by a different protein subunit. 
Leucine zipper proteins thus bind to DNA 
as dimers, gripping the double helix like a 
clothespin on a clothesline (Movie 8.2). 

Figure 8-6 A cluster of bacterial genes 
can be transcribed from a single 
promoter. Each of these five genes encodes 
a different enzyme; all of the enzymes 
are needed to synthesize the amino acid 
tryptophan. The genes are transcribed as a 
single mRNA molecule, a feature that allows 
their expression to be coordinated. Clusters 
of genes transcribed as a single mRNA 
molecule are common in bacteria. Each of 
these clusters is called an operon because 
its expression is controlled by a regulatory 
DNA sequence called the operator (green), 
situated within the promoter. The yellow 
blocks in the promoter represent DNA 
sequences that bind RNA polymerase. 

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CHAPTER 8 Control of Gene Expression 

promoter sequences 

-60 -35 Í8SSÍ*?j-10 +1 +20 



Figure 8-7 Genes can be switched off by repressor proteins. If the concentration of tryptophan ¡nside a 
bacterium ¡s low ( left ), RNA polymerase (b/ue) binds to the promoter and transcribes the five genes of the tryptophan 
operon. However, if the concentration of tryptophan ¡s high (ríght), the repressor protein (dark green) becomes active 
and binds to the operator (light green), where ¡t blocks the binding of RNA polymerase to the promoter. Whenever 
the concentration of intracellular tryptophan drops, the repressor falls off the DNA, allowing the polymerase to 
again transcribe the operon. The promoter contains two key blocks of DNA sequence information, the -35 and -10 
regions, highlighted in yellow, which are recognized by RNA polymerase (see Figure 7-10). The complete operon is 
shown in Figure 8-6. 

Repressors Turn Genes Off and Activators Turn Them On 

The tryptophan repressor, as its ñame suggests, is a transcriptional 
repressor protein: in its active form, it switches genes off, or represses 
them. Some bacterial transcription regulators do the opposite: they switch 
genes on, or actívate them. These transcriptional activator proteins 
work on promoters that—in contrast to the promoter for the tryptophan 
operon—are only marginally able to bind and position RNA polymerase 
on their own. However, these poorly functioning promoters can be made 
fully functional by activator proteins that bind nearby and contact the 
RNA polymerase to help it initiate transcription (Figure 8-8). 

Like the tryptophan repressor, activator proteins often have to interact 
with a second molecule to be able to bind DNA. For example, the bacte¬ 
rial activator protein CAP has to bind cyclic AMP (cAMP) before it can 
bind to DNA (see Figure 4-19). Genes activated by CAP are switched on 
in response to an increase in intracellular cAMP concentration, which 
rises when glucose, the bacterium's preferred carbón source, is no longer 
available; as a result, CAP drives the production of enzymes that allow 
the bacterium to digest other sugars. 

Figure 8-8 Genes can be switched on by 
activator proteins. An activator protein 
binds to a regulatory sequence on the DNA 
and then ¡nteracts with the RNA polymerase 
to help it initiate transcription. Without 
the activator, the promoter fails to initiate 
transcription efficiently. In bacteria, the 
binding of the activator to DNA is often 
controlled by the interaction of a metabolite 
or other small molecule (red triangle) with 
the activator protein. The L ac operon works 
in this manner, as we discuss shortly. 

An Activator and a Repressor Control the Lac Operon 

In many instances, the activity of a single promoter is controlled by two 
different transcription regulators. The Lac operon in E. coli, for example, 

RNA polymerase 


binding site 
for activator 

LibertadDigital \ 2015 

How Transcriptional Switches Work 269 

is controlled by both the Lac repressor and the CAP activator that we 
just discussed. The Lac operon encodes proteins required to import and 
digest the disaccharide lactose. In the absence of glucose, the bacterium 
makes cAMP, which activates CAP to switch on genes that allow the cell 
to utilize alternative sources of carbón—including lactose. It would be 
wasteful, however, for CAP to induce expression of the Lac operon if lac¬ 
tose itself were not present. Thus the Lac repressor shuts off the operon 
in the absence of lactose. This arrangement enables the control región 
of the Lac operon to intégrate two different signáis, so that the operon 
is highly expressed only when two conditions are met: glucose must be 
absent and lactose must be present (Figure 8-9). This genetic Circuit thus 
behaves much like a switch that carries out a logic operation in a Compu¬ 
ter. When lactose is present AND glucose is absent, the cell executes the 
appropriate program—in this case, transcription of the genes that permit 
the uptake and utilization of lactose. 

The elegant logic of the Lac operon first attracted the attention of biolo- 
gists more than 50 years ago. The molecular basis of the switch in E. coli 
was uncovered by a combination of genetics and biochemistry, provid- 
ing the first insight into how transcription is controlled. In a eukaiyotic 
cell, similar transcription regulatory devices are combined to generate 
increasingly complex circuits, including those that enable a fertilized egg 
to form the tissues and organs of a multicellular organism. 


Bacterial cells can take up the 
amino acid tryptophan (Trp) from 
their surroundings, or if there is an 
insufficient external supply they can 
synthesize tryptophan from other 
small molecules. The Trp repressor is 
a transcription regulator that shuts 
off the transcription of genes that 
code for the enzymes required for 
the synthesis of tryptophan (see 
Figure 8-7). 

A. What would happen to the 
regulation of the tryptophan operon 
in cells that express a mutant form 
of the tryptophan repressor that 

(1) cannot bind to DNA, (2) cannot 
bind tryptophan, or (3) binds 
to DNA even in the absence of 

B. What would happen in 
scenarios (1), (2), and (3) if the 
cells, in addition, produced normal 
tryptophan repressor protein from a 
second, normal gene? 


CAP- polymerase- start of transcription 
binding binding site I 
site (promoter) 

operator LacZ gene 

nudeotide pairs 






RNA polymerase 

Figure 8-9 The Lac operon is controlled by two transcription regulators, 
the Lac repressor and CAP. When lactose is absent, the Lac repressor binds 
to the Lac operator and shuts off expression of the operon. Addition of lactose 
¡ncreases the intracellular concentraron of a related compound, allolactose; 
allolactose binds to the Lac repressor, causing itto undergo a conformational 
change that releases its grip on the operator DNA (not shown). When glucose is 
absent, cyclic AMP (red triangle) is produced by the cell, and CAP binds to DNA. 
LacZ, the first gene of the operon, encodes the enzyme |3-galactos¡dase, which 
breaks down lactose to galactose and glucose. 

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CHAPTER 8 Control of Gene Expression 


Explain how DNA-binding proteins 
can make sequence-specific contacts 
to a double-stranded DNA molecule 
without breaking the hydrogen 
bonds that hold the bases together. 
Indícate how, through such contacts, 
a protein can distinguish a T-A from 
a C-G pair. Indícate the parts of the 
nucleotide base palrs that could 
form noncovalent interactions— 
hydrogen bonds, electrostatic 
attractions, or hydrophoblc 
interactions (see Panel 2-7, 
pp. 78-79)—with a DNA-binding 
protein. The structures of all the 
base pairs ¡n DNA are given in 
Figure 5-6. 

Figure 8-10 In eukaryotes, gene 
activation can occur at a distance. 

An activator protein bound to a distant 
enhancer attracts RNA polymerase 
and general transcription factors to the 
promoter. Looping of the ¡ntervening DNA 
permits contact between the activator and 
the transcription initiation complex bound 
to the promoter. In the case shown here, 
a large protein complex called Mediator 
serves as a go-between. The broken stretch 
of DNA signifies that the length of DNA 
between the enhancer and the start of 
transcription varíes, sometimes reaching 
tens ofthousands of nucleotide pairs in 
length. The TATA box is a DNA recognition 
sequence for the first general transcription 
factor that binds to the promoter (see 
Figure 7-12). 

Eukaryotic Transcription Regulators Control Gene 
Expression from a Distance 

Eukaryotes, too, use transcription regulators—both activators and 
repressors—to regúlate the expression of their genes. The DNA sites to 
which eukaryotic gene activators bind are termed enhancers, because 
their presence dramatically enhances the rate of transcription. It was 
surprising to biologists when, in 1979, it was discovered that these acti¬ 
vator proteins could enhance transcription even when they are bound 
thousands of nucleotide pairs away from a gene's promoter. They also 
work when bound either upstream or downstream from the gene. These 
observations raised several questions. How do enhancer sequences and 
the proteins bound to them function over such long distances? How do 
they communicate with the promoter? 

Many models for this "action at a distance" have been proposed, but the 
simplest of these seems to apply in most cases. The DNA between the 
enhancer and the promoter loops out to allow eukaryotic activator pro¬ 
teins to influence directly events that take place at the promoter (Figure 
8-10). The DNA thus acts as a tether, allowing a protein that is bound 
to an enhancer—even one that is thousands of nucleotide pairs away— 
to interact with the proteins in the vicinity of the promoter—including 
RNA polymerase and the general transcription factors (see Figure 7-12). 
Often, additional proteins serve to link the distantly bound transcription 
regulators to these proteins at the promoter; the most important of these 
regulators is a large complex of proteins known as Mediator (see Figure 
8-10). One of the ways in which these proteins function is by aiding the 
assembly of the general transcription factors and RNA polymerase to 
form a large transcription complex at the promoter. Eukaryotic repressor 
proteins do the opposite: they decrease transcription by preventing the 
assembly of the same protein complex. 

In addition to promoting—or repressing—the assembly of a transcription 
initiation complex directly, eukaryotic transcription regulators have an 
additional mechanism of action: they attract proteins that modify chro- 
matin structure and thereby affect the accessibility of the promoter to the 
general transcription factors and RNA polymerase, as we discuss next. 


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How Transcriptional Switches Work 


Eukaryotic Transcription Regulators Help Initiate 
Transcription by Recruiting Chromatin-Modifying Proteins 

Initiation of transcription in eukaryotic cells must also take into account 
the packaging of DNA into chromosomes. As discussed in Chapter 5, 
eukaryotic DNA is packed into nucleosomes, which, in turn, are folded 
into higher-order structures. How do transcription regulators, general 
transcription factors, and RNA polymerase gain access to such DNA? 
Nucleosomes can inhibit the initiation of transcription if they are posi- 
tioned over a promoter, because they physically block the assembly of 
the general transcription factors or RNA polymerase on the promoter. 
Such chromatin packaging may have evolved in part to prevent leaky 
gene expression by blocking the initiation of transcription in the absence 
of the proper activator proteins. 

In eukaryotic cells, activator and repressor proteins exploit chromatin 
structure to help turn genes on and off. As we saw in Chapter 5, chroma¬ 
tin structure can be altered by chromatin-remodeling complexes and by 
enzymes that covalently modify the histone proteins that form the core of 
the nucleosome (see Figures 5-26 and 5-27). Many gene activators take 
advantage of these mechanisms by recruiting such chromatin-modifying 
proteins to promoters. For example, the recruitment of histone acetyl- 
transferases promotes the attachment of acetyl groups to selected lysines 
in the tail of histone proteins. This modiflcation alters chromatin struc¬ 
ture, allowing greater accessibility to the underlying DNA; moreover, the 
acetyl groups themselves attract proteins that promote transcription, 
including some of the general transcription factors (Figure 8-11). 

Likewise, gene repressor proteins can modify chromatin in ways that 
reduce the efficiency of transcription initiation. For example, many repres- 
sors attract histone deacetylases—e nzymes that remove the acetyl groups 
from histone tails, thereby reversing the positive effects that acetylation 
has on transcription initiation. Although some eukaryotic repressor pro¬ 
teins work on a gene-by-gene basis, others can orchestrate the formation 
of large swathes of transcriptionally inactive chromatin containing many 


Some transcription regulators bind 
to DNA and cause the double helix 
to bend at a sharp angle. Such 
"bending proteins" can stimulate 
the initiation of transcription 
without contacting either the RNA 
polymerase, any of the general 
transcription factors, or any other 
transcription regulators. Can you 
devise a plausible explanation for 
how these proteins might work 
to modulate transcription? Draw 
a diagram that ¡llustrates your 

histone transcription regulator 

Figure 8-11 Eukaryotic transcriptional 
activators can recruit chromatin- 
modifying proteins to help initiate gene 
transcription. On the right, chromatin- 
remodeling complexes renderthe DNA 
packaged in chromatin more accessible to 
other proteins in the cell, including those 
required for transcription initiation; notice, 
for example, the increased exposure of the 
TATA box. On the left, the recruitment of 
histone-modifying enzymes such as histone 
acetyltransferases adds acetyl groups to 
specific histones, which can then serve as 
binding sites for proteins that stimulate 
transcription initiation (not shown). 

LibertadDigital \ 2015 

272 CHAPTER 8 Control of Gene Expression 

genes. As discussed in Chapter 5, these transcription-resistant regions of 
DNA inelude the heterochromatin found in interphase chromosomes and 
the inactive X chromosome in the cells of female mammals. 


All cells musí be able to switch genes on and off in response to signáis in 
their environment. But the cells of multicellular organisms have evolved 
this capacity to an extreme degree and in highly specialized ways to form 
organized arrays of differentiated cell types. In particular, once a cell in a 
multicellular organism becomes committed to differentiate into a specific 
cell type, the choice of fate is generally maintained through subsequent 
cell divisions. This means that the changes in gene expression, which are 
often triggered by a transient signal, musí be remembered by the cell. This 
phenomenon of cell memoiy is a prerequisite for the creation of organ¬ 
ized tissues and for the maintenance of stably differentiated cell types. 
In contrast, the simplest changes in gene expression in both eukaryotes 
and bacteria are often only transient; the tryptophan repressor, for exam- 
ple, switches off the tryptophan operon in bacteria only in the presence 
of tryptophan; as soon as the amino acid is removed from the médium, 
the genes switch back on, and the descendants of the cell will have no 
memory that their ancestors had been exposed to tryptophan. 

In this section, we discuss some of the special features of transcriptional 
regulation that are found in multicellular organisms. Our focus will be 
on how these mechanisms create and maintain the specialized cell types 
that give a worm, a fly, or a human its distinctive characteristics. 

Eukaryotic Genes Are Controlled by Combinations of 
Transcription Regulators 

Because eukaryotic transcription regulators can control transcription ini- 
tiation when bound to DNA many base pairs away from the promoter, the 
nucleotide sequences that control the expression of a gene can be spread 
over long stretches of DNA. In animáis and plants, it is not unusual to 
find the regulatory DNA sequences of a gene dotted over tens of thou- 
sands of nucleotide pairs, although much of the intervening DNA serves 
as "spacer" sequence and is not directly recognized by the transcription 

So far in this chapter, we have treated transcription regulators as though 
each functions individually to tum a gene on or off. While this idea holds 
true for many simple bacterial activators and repressors, most eukaryotic 
transcription regulators work as part of a "committee" of regulatory pro- 
teins, all of which are necessary to express the gene in the right place, in 
the right cell type, in response to the right conditions, at the right time, 
and in the required amount. 

The term combinatorial control refers to the way that groups of tran¬ 
scription regulators work together to determine the expression of a single 
gene. We saw a simple example of such regulation by múltiple regula¬ 
tors when we discussed the bacterial Lac operon (see Figure 8-9). In 
eukaryotes, the regulatory inputs have been amplified, and a typical gene 
is controlled by dozens of transcription regulators. These help assem- 
ble chromatin-remodeling complexes, histone-modifying enzymes, 
RNA polymerase, and general transcription factors via the multiprotein 
Mediator complex (Figure 8-12). In many cases, both repressors and 
activators will be present in the same complex; how the cell integrates 
the effeets of all of these proteins to determine the final level of gene 

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The Molecular Mechanlsms That Create Speclallzed Cell Types 


regulatory DNA sequences 


Figure 8-12 Transcription regulators 
work together as a "committee" to 
control the expression of a eukaryotic 
gene. Whereas the general transcription 
factors that assemble at the promoter 
are the same for all genes transcrlbed by 
RNA polymerase (see Figure 7-12), the 
transcription regulators and the locations 
of their DNA binding sites relative to the 
promoters are different for different genes. 
These regulators, along with chromatin- 
modifying proteins, are assembled atthe 
promoter by the Mediator. The effects of 
múltiple transcription regulators combine 
to determine the final rate of transcription 

expression is only now beginning to be understood. An example of such 
a complex regulatory system—one that participates in the develop- 
ment of a fruit fly from a fertilized egg—is described in How We Know, 

The Expression of Different Genes Can Be Coordinated by 
a Single Protein 

In addition to being able to switch individual genes on and off, all cells— 
whether prokaiyote or eukaryote—need to coordínate the expression of 
different genes. When a eukaryotic cell receives a signal to divide, for 
example, a number of hitherto unexpressed genes are tumed on together 
to set in motion the events that lead eventually to cell división (discussed 
in Chapter 18). As discussed earlier, one way in which bacteria coordí¬ 
nate the expression of a set of genes is by having them clustered together 
in an operon under the control of a single promoter (see Figure 8-6). 
Such clustering is not seen in eukaryotic cells, where each gene is tran- 
scribed and regulated individually. So how do these cells coordínate gene 
expression? In particular, given that a eukaryotic cell uses a committee 
of transcription regulators to control each of its genes, how can it rapidly 
and decisively switch whole groups of genes on or off? 

The answer is that even though control of gene expression is combinato- 
rial, the effect of a single transcription regulator can still be decisive in 
switching any particular gene on or off, simply by completing the com- 
bination needed to actívate or repress that gene. This is like dialing in 
the final number of a combination lock: the lock will spring open if the 
other numbers have been previously entered. Just as the same number 
can complete the combination for different locks, the same protein can 
complete the combination for several different genes. As long as differ¬ 
ent genes contain regulatory DNA sequences that are recognized by the 
same transcription regulator, they can be switched on or off together, as 
a coordinated unit. 

An example of such coordinated regulation in humans is seen with the 
cortisol receptor protein. In order to bind to regulatory sites in DNA, this 

LibertadDigital \ 2015 




The ability to regúlate gene expression is crucial to 
the proper development of a multicellular organism 
from a fertilized egg to a fertile adult. Beginning at the 
earliest moments in development, a succession of tran- 
scriptional programs guides the differential expression 
of genes that allows an animal to form a proper body 
plan—helping to distinguish its back from its belly, and 
its head from its tail. These programs ultimately direct 
the correct placement of a wing or a leg, a mouth or an 
anus, a neuron or a sex cell. 

A central challenge in development, then, is to under- 
stand how an organism generates these pattems of 
gene expression, which are laid down within hours of 
fertilization. Among the most important genes involved 
in these early stages of development are those that 
encode transcription regulators. By interacting with dif- 
ferent regulatory DNA sequences, these proteins instruct 
eveiy cell in the embiyo to switch on the genes that are 
appropriate for that cell at each time point during devel¬ 
opment. How can a protein binding to a piece of DNA 
help direct the development of a complex multicellular 
organism? To see how we can address that large ques- 
tion, we review the story of Eve. 

Seeing Eve 

Even-skipped—Eve, for short—is a gene whose expres¬ 
sion plays an important part in the development of the 
Drosophila embryo. If this gene is inactivated by muta- 
tion, many parts of the embryo fail to form and the fly 
larva dies early in development. But Eve is not expressed 
uniformly throughout the embryo. Instead, the Eve pro¬ 
tein is produced in a striking series of seven neat stripes, 
each of which occupies a veiy precise position along the 
length of the embryo. These seven stripes correspond to 
seven of the fourteen segments that define the body plan 
of the fly—three for the head, three for the thorax, and 
eight for the abdomen. 

This pattern never varíes: Eve can be found in the veiy 
same places in every Drosophila embiyo (see Figure 
8-13B). How can the expression of a gene be regulated 
with such spatial precisión—such that one cell will pro¬ 
duce a protein while a neighboring cell does not? To find 
out, researchers took a trip upstream. 

Dissecting the DNA 

As we have seen in this chapter, regulatory DNA 
sequences control which cells in an organism will 
express a particular gene, and at what point during 
development that gene will be turned on. In eukaryotes, 

these regulatory sequences are frequently located 
upstream of the gene itself. One way to lócate a regu¬ 
latory DNA sequence—and study how it operates—is 
to remove a piece of DNA from the región upstream 
of a gene of interest and inserí that DNA upstream of 
a repórter gene—one that encodes a protein with an 
activity that is easy to monitor experimentally. If the 
piece of DNA contains a regulatory sequence, it will 
drive the expression of the repórter gene. When this 
patchwork piece of DNA is subsequently introduced into 
a cell or organism, the repórter gene will be expressed 
in the same cells and tissues that normally express the 
gene from which the regulatory sequence was derived 
(see Figure 10-31). 

By excising various segments of the DNA sequences 
upstream of Eve, and coupling them to a repórter gene, 
researchers found that the expression of the gene is 
controlled by a series of seven regulatory modules— 
each of which specifies a single stripe of Eve expression. 
In this way, researchers identified, for example, a sin¬ 
gle segment of regulatory DNA that specifies stripe 2. 
They could excise this regulatory segment, link it to a 
repórter gene, and introduce the resulting DNA segment 
into the fly. When they examined embryos that carried 
this engineered DNA, they found that the repórter gene 
is expressed in the precise position of stripe 2 (Figure 
8-13) . Similar experiments revealed the existence of six 
other regulatory modules, one for each of the other Eve 

The next question is: How does each of these seven reg- 
ulatory segments direct the formation of a single stripe 
in a specific position? The answer, researchers found, 
is that each segment contains a unique combination 
of regulatory sequences that bind different combina- 
tions of transcription regulators. These regulators, like 
Eve itself, are distributed in unique patterns within the 
embryo—some toward the head, some toward the rear, 
some in the middle. 

The regulatory segment that defines stripe 2, for 
example, contains regulatory DNA sequences for four 
transcription regulators: two that actívate Eve transcrip¬ 
tion and two that repress it (Figure 8-14). In the narrow 
band of tissue that constitutes stripe 2, it just so happens 
the repressor proteins are not present—so the Eve gene 
is expressed; in the bands of tissue on either side of the 
stripe, the repressors keep Eve quiet. And so a stripe is 

The regulatory segments controlling the other stripes 
are thought to function along similar Unes; each regu¬ 
latory segment reads "positional information" provided 

'JbertadDigital \ 2015 

The Molecular Mechanlsms That Create Speclallzed Cell Types 




\ l M / v 

Eve regulatory segments ^ 


TATA Eve gene 

stripe 2 TATA LacZ gene 

reguiatory box 





Figure 8-13 An experimental approach that involves the use of a repórter gene reveáis the modular construction 

of the Eve gene regulatory región. (A) Expresslon of the Eve gene ¡s controlled by a serles of regulatory segments 
( orange ) that directthe productlon of Eve proteln ¡n stripes along the embryo. (B) Embryos stalned with antibodles to 
the Eve proteln show the seven characteristlc stripes of Eve expresslon. (C) In the laboratory, the regulatory segment that 
dlrects the formation of stripe 2 can be excised from the DNAshown in part A and ¡nserted upstream of the E. coli LacZ 
gene, which encodes the enzyme